U.S. patent number 10,605,766 [Application Number 14/798,762] was granted by the patent office on 2020-03-31 for biochemically activated electronic device.
This patent grant is currently assigned to ILLUMINA, INC.. The grantee listed for this patent is ILLUMINA, INC.. Invention is credited to Jingwei Bai, Boyan Boyanov, Cheng-Yao Chen, Kevin L. Gunderson, Jeffrey G. Mandell, Michel Perbost.
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United States Patent |
10,605,766 |
Boyanov , et al. |
March 31, 2020 |
Biochemically activated electronic device
Abstract
A method of nucleic acid sequencing. The method can include the
steps of (a) providing a polymerase tethered to a solid support
charge sensor; (b) providing one or more nucleotides, whereby the
presence of the nucleotide can be detected by the charge sensor;
and (c) detecting incorporation of the nucleotide into a nascent
strand complementary to a template nucleic acid.
Inventors: |
Boyanov; Boyan (San Diego,
CA), Mandell; Jeffrey G. (La Jolla, CA), Bai; Jingwei
(San Diego, CA), Gunderson; Kevin L. (Encinitas, CA),
Chen; Cheng-Yao (San Diego, CA), Perbost; Michel (San
Diego, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, INC. |
San Diego |
CA |
US |
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Assignee: |
ILLUMINA, INC. (San Diego,
CA)
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Family
ID: |
53783324 |
Appl.
No.: |
14/798,762 |
Filed: |
July 14, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160017416 A1 |
Jan 21, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62171523 |
Jun 5, 2015 |
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62161709 |
May 14, 2015 |
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62069198 |
Oct 27, 2014 |
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62024856 |
Jul 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
27/4145 (20130101); C12Q 1/6825 (20130101); C12Q
1/6869 (20130101); C12Q 1/6869 (20130101); C12Q
2525/197 (20130101); C12Q 2535/122 (20130101); C12Q
2563/113 (20130101); C12Q 2563/116 (20130101); C12Q
2565/107 (20130101); C12Q 2565/607 (20130101); C12Q
2565/629 (20130101); C12Q 1/6869 (20130101); C12Q
2521/101 (20130101); C12Q 2565/607 (20130101); C12Q
1/6825 (20130101); C12Q 2521/101 (20130101); C12Q
2523/307 (20130101); C12Q 2523/319 (20130101); C12Q
2525/197 (20130101); B82Y 15/00 (20130101); B01J
2219/00653 (20130101); B01J 2219/00722 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); C12Q 1/6825 (20180101); G01N
27/414 (20060101); C12Q 1/6869 (20180101); B82Y
15/00 (20110101) |
References Cited
[Referenced By]
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WO |
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0125480 |
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Apr 2001 |
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WO |
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Mar 2004 |
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WO |
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2007/123744 |
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Nov 2007 |
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WO |
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2008/076406 |
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Jun 2008 |
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WO |
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2008/107014 |
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Sep 2008 |
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WO |
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2010/068884 |
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Jun 2010 |
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WO |
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2012/116191 |
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Aug 2012 |
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WO |
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2013/056241 |
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Apr 2013 |
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WO |
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2013/154999 |
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Oct 2013 |
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WO |
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Primary Examiner: Sisson; Bradley L.
Attorney, Agent or Firm: Illumina, Inc.
Parent Case Text
This application is based on, and claims the benefit of, U.S.
Provisional Application No. 62/171,523, filed Jun. 5, 2015; U.S.
Provisional Application No. 62/161,709, filed May 14, 2015; U.S.
Provisional Application No. 62/069,198, filed Oct. 27, 2014; and
U.S. Provisional Application No. 62/024,856, filed Jul. 15, 2014,
each of which is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method of nucleic acid sequencing, comprising: (a) contacting
a nucleic acid molecule to be sequenced with a polymerase attached
to a solid support charge sensor with an oligonucleotide tether;
(b) contacting the nucleic acid molecule and the polymerase with
different nucleotide types, wherein each of the different
nucleotide types is respectively attached to an oligonucleotide
capable of hybridizing to a complementary region of the
oligonucleotide tether and includes a unique label that can be
detected by the solid support charge sensor when the label is in
proximity of a screening length of the charge sensor wherein the
polymerase catalyzes incorporation of one of the different
nucleotide types to a nascent nucleic acid strand complementary to
the nucleic acid molecule and wherein the oligonucleotide of the
one of the different nucleotide types hybridizes to the
complementary region of the oligonucleotide tether and brings the
unique label within proximity of the screening length; and (c)
detecting a distinguishable signal using the charge sensor in
response to the incorporation of the one of the different
nucleotide types into the nascent nucleic acid strand complementary
to the nucleic acid molecule to be sequenced.
2. The method of claim 1, wherein a phosphate of the incorporated
one of the different nucleotide types is cleaved by the polymerase
after incorporation, and wherein the oligonucleotide of the
incorporated one of the different nucleotide types dissociates from
the oligonucleotide tether.
3. The method of claim 2, wherein the polymerase catalyzes
incorporation of a next of the different nucleotide types to the
nascent nucleic acid strand complementary to the nucleic acid
molecule, wherein the oligonucleotide of the next of the different
nucleotide types hybridizes to its complementary region of the
oligonucleotide tether and brings its unique label within proximity
of the screening length, and wherein the method further comprises
detecting a next distinguishable signal using the charge sensor in
response to the incorporation of the next of the different
nucleotide types into the nascent nucleic acid strand complementary
to the nucleic acid molecule to be sequenced.
4. The method of claim 1, wherein the different nucleotide types
comprise four different types of nucleotides, wherein each of the
four different types of nucleotides comprises a unique charge
label.
5. The method of claim 1, wherein the solid support charge sensor
comprises a nanowire field-effect transistor (FET) or a carbon
nanotube field-effect transistor (FET).
6. The method of claim 1, wherein the solid support charge sensor
is one of a plurality of charge sensors in an array and wherein the
polymerase is one of a plurality of polymerases, wherein each of
the plurality of charge sensors is tethered to a respective one of
the plurality of polymerases, and step (c) comprises detecting
distinguishable signals at each of the plurality of charge sensors
in response to incorporation of respective nucleotide types into
respective nascent complementary strands at each polymerase.
7. The method of claim 1, wherein each of the different nucleotide
types is a reversibly terminated nucleotide, wherein the polymerase
lacks a 3' to 5' exonuclease function, and wherein the method
further comprises adding a deblocking agent prior to performing a
subsequent sequencing cycle.
8. The method of claim 7, wherein the polymerase comprises the
large fragment of Bsu DNA polymerase I.
9. The method of claim 8, wherein the large fragment of Bsu DNA
polymerase I is modified to include residues from the O-helix
finger domain of Klenow fragment.
10. The method of claim 1, wherein the solid support charge sensor
comprises a variable width including a region of substantially
narrower width, wherein the region of substantially narrower width
is 10% to 75% of a diameter of larger width regions of the solid
support charge sensor.
11. The method of claim 10, wherein the polymerase is tethered to
the solid support charge sensor by attachment to the region of
substantially narrower width.
12. The method of claim 1, wherein the oligonucleotide tether
comprises native ribonucleotides or analogs thereof.
13. The method of claim 12, wherein the oligonucleotide capable of
hybridizing to the complementary region of the oligonucleotide
tether is the unique label, and wherein the unique label comprises
native ribonucleotides or analogs thereof.
14. The method of claim 1, wherein the oligonucleotide tether
comprises native deoxyribonucleotides or analogs thereof.
15. The method of claim 14, wherein the oligonucleotide tether
comprises native deoxyribonucleotides or analogs thereof, wherein
the oligonucleotide capable of hybridizing to the complementary
region of the oligonucleotide tether is the unique label, and
wherein the unique label comprises native deoxyribonucleotides or
analogs thereof.
16. The method of claim 1, wherein the different nucleotide types
comprise reversible terminator moieties; wherein the incorporation
of the one of the different nucleotide types incorporates a
reversibly terminated nucleotide into the nascent nucleic acid
strand; and wherein the method further comprises (d) adding a
deblocking agent to render the nascent nucleic acid strand capable
of further incorporation of a next nucleotide; and (e) repeating
steps (b) through (d) to obtain a sequence of the nucleic acid
molecule to be sequenced.
17. The method of claim 1, wherein the unique label of the one of
the different nucleotide types comprises a unique charge tag that
is detected by the charge sensor during incorporation of the one
nucleotide type into the nascent nucleic acid strand complementary
to the nucleic acid molecule to be sequenced.
18. The method of claim 1, wherein the oligonucleotide of each of
the different nucleotide types is the unique label, wherein the
oligonucleotide of each of the different nucleotide types
hybridizes to a different location of the oligonucleotide
tether.
19. The method of claim 1, wherein the polymerase is in proximity
of less than 10 nm to the charge sensor.
20. A method of detecting incorporation of nucleotides, comprising:
(a) contacting a nucleic acid molecule to be sequenced with a
polymerase, attached to a solid support charge sensor with an
oligonucleotide tether; (b) contacting the nucleic acid molecule
and the polymerase with four different nucleotide types, wherein
each of the four different nucleotide types is respectively
attached to an oligonucleotide capable of hybridizing to a
complementary region of the oligonucleotide tether and includes a
charge unique label that can be detected by the solid support
charge sensor when the unique charge label is in proximity of a
screening length of the charge sensor, wherein the polymerase
catalyzes incorporation of one of the four different nucleotide
types to a nascent nucleic acid strand complementary to the nucleic
acid molecule and wherein the oligonucleotide of the one of the
four different nucleotide types hybridizes to the complementary
region of the oligonucleotide tether and brings the unique charge
label within proximity of the screening length; (c) detecting a
distinguishable signal of the unique charge label using the charge
sensor; and (d) identifying the incorporated one of the four
different nucleotide types by the distinguishable signal.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Aug. 25,
2015, is named IP-1285-US_SL.txt and is 15,955 bytes in size.
BACKGROUND
This disclosure relates generally to biosensor-based detection, and
more specifically to biosensors that can be used for nucleic acid
sequencing.
Currently available commercial platforms for sequencing DNA are
relatively costly. The majority of these platforms use a
`sequencing-by-synthesis` approach, so called because DNA polymers
are synthesized while detecting the addition of each monomer (i.e.
nucleotide) to the growing polymer structure. Because a template
DNA strand strictly directs synthesis of a new DNA polymer, one can
infer the sequence of the template DNA from the series of
nucleotide monomers that were added to the growing strand during
the synthesis. Monitoring the reaction uses relatively expensive
hardware such as lasers, detection optics and complex fluid
delivery systems. The most successful commercial platforms to date
also require expensive reagents and hardware to amplify the DNA
templates before sequencing-by-synthesis can even begin. The
complexity and expense of these platforms has hindered their use in
some clinical and research contexts where there is a clear need for
DNA sequencing technology.
Thus, there exists a need for improvements to nucleic acid
sequencing platforms to make them more cost effective, rapid and
convenient. The present disclosure addresses this need and provides
other advantages as well.
BRIEF SUMMARY
The present disclosure provides a first method of nucleic acid
sequencing. The method can include the steps of (a) providing a
polymerase tethered to a solid support charge sensor; (b) providing
one or more labeled nucleotides, whereby the presence of the label
can be detected by the charge sensor when the label is in proximity
to the charge sensor; and (c) detecting incorporation of the
labeled nucleotide into a nascent strand complementary to a
template nucleic acid.
The present disclosure also provides a method for attaching
reaction components to charge sensors. The method can include the
steps of (a) providing a solid support including a plurality of
charge sensors, wherein each of the charge sensors has a capacity
to attach a plurality of reaction components; (b) providing a fluid
containing a plurality of reaction components of a particular type;
and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; and (iii) reaction components of the
particular type from the fluid attach to the charge sensors under
conditions that result in a solid support where each of the charge
sensors is attached to a single one of the reaction components.
In some embodiments the method for attaching reaction components to
charge sensors can include the steps of (a) providing a solid
support including a plurality of charge sensors, wherein each of
the charge sensors has a capacity to attach a plurality of reaction
components; (b) providing a fluid containing a plurality of
reaction components of a particular type, wherein each of the
reaction components of the particular type is bound to a repellant
moiety; and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; (iii) reaction components from the fluid
attach to the charge sensors, and (iv) the repellant moiety bound
to each of the reaction components prevents more than one of the
reaction components in the plurality of reaction components from
attaching to each of the charge sensors.
A method for attaching reaction components to charge sensors can
include the steps of (a) providing a solid support including a
plurality of charge sensors, wherein each of the charge sensors has
a capacity to attach a plurality of reaction components; (b)
providing a fluid containing a plurality of reaction components of
a particular type; (c) contacting the solid support with the fluid
under conditions wherein (i) the plurality of reaction components
of the particular type are in fluid communication with the
plurality of charge sensors, (ii) a greater number of reaction
components of the particular type is in the fluid than the number
of charge sensors on the solid support; and (iii) reaction
components of the particular type from the fluid attach to the
charge sensors, thereby forming modified charge sensors that are
attached to multiple reaction components of the particular type
from the fluid; and (d) removing one or more of the reaction
components of the particular type from each of the modified charge
sensors to leave a single one of the reaction components of the
particular type attached to each of the modified charge
sensors.
The present disclosure provides a method of detecting a nucleotide.
The method can include the steps of (a) providing a nucleotide
binding protein (e.g. a polymerase) tethered to a solid support
charge sensor; (b) providing one or more labeled nucleotides,
whereby the presence of the label can be detected by the charge
sensor when the label is in proximity to the charge sensor; and (c)
detecting binding of the labeled nucleotide to the protein using
the charge sensor.
In particular embodiments, a method of nucleic acid sequencing can
be performed by (a) providing a polymerase tethered to a solid
support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting incorporation of the labeled nucleotide
into a nascent strand complementary to a template nucleic acid
using the charge sensor.
A method of nucleic acid sequencing provided by the present
disclosure can include the steps of (a) providing a polymerase
tethered to a solid support charge sensor; (b) providing one or
more labeled nucleotides, whereby the presence of the label can be
detected by the charge sensor when the label is in proximity to the
charge sensor, wherein the one or more labeled nucleotides have
reversible terminator moieties; (c) detecting incorporation of the
one or more labeled nucleotides into a nascent strand complementary
to a template nucleic acid using the charge sensor, thereby forming
a reversibly terminated nascent strand; (d) modifying the
reversible terminated nascent strand to render the nascent strand
capable of further incorporation of nucleotide; and (e) repeating
(b) through (d) to obtain a sequence of the template nucleic
acid.
Also provided is a method of nucleic acid sequencing that includes
the steps of (a) providing a polymerase tethered to a solid support
charge sensor; (b) contacting the polymerase with a template
nucleic acid and one or more different nucleotide types under
conditions wherein the polymerase catalyzes addition of the one or
more nucleotide types to form a nucleic acid complement of the
nucleic acid template, and wherein the addition of one or more
different nucleotide types produces a conformational signal change
from the polymerase that is detected by the charge sensor; (c)
detecting a change in the signal from the polymerase using the
charge sensor; and (d) determining the rate, polarity, amplitude or
time duration for the change in the signal for the addition of the
one or more different nucleotide type, thereby determining a
sequence of nucleotides for the template nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a polymerase attached to a charge sensor via a
tether.
FIG. 2 shows polymerases attached to charge sensors via nucleic
acid tethers and bound to nucleotides that can be distinguished
based on charge or proximity to the sensor.
FIG. 3 shows polymerases attached to charge sensors via nucleic
acid tethers and bound to nucleotides that can be distinguished
based on charge.
FIG. 4 shows a polymerase tethered to a charge sensor, wherein the
charge sensor is also attached to a plurality of oligonucleotides
capable of binding to labels on nucleotides.
FIG. 5 shows a nucleotide label having two negatively charged
oxygens at the end of an oligonucleotide moiety of the label.
FIG. 6 shows charge tags that can be detected using a charge
sensor.
FIG. 7 shows aligned sequences (SEQ ID NOS 1-3, respectively, in
order of appearance) of Klenow fragment (Pollxxo) and Bsu-LF
(Bsuxxx1), in which the location of the O-helix finger domain of
Klenow fragment and the aligned location in Bsu-LF is boxed. Also
shown in the inset at the right is a model showing the three
dimensional locations of several residues of the Klenow fragment
O-helix finger juxtaposed with a model of a nanotube.
FIG. 8 shows a charge sensor having a pinched region and a
polymerase tethered to the charge sensor at the pinched region. The
polymerase is complexed with a template strand and nascent strand
of a nucleic acid.
FIG. 9 shows a diagrammatic representation for a method of
introducing a pinch into silicon using local oxidation.
FIG. 10 shows a charge sensor that is attached to a polymerase
(Pol) via a tether having a nucleic acid sequence (generically
represented as a sequence of 10 Ns). The polymerase is complexed to
a target nucleic acid and binding sites for labels associated with
four different nucleotides (ATP, GTP, CTP and TTP, respectively)
are indicated.
FIG. 11 shows a charge sensor that is attached to a polymerase
(Pol) via a tether having a nucleic acid sequence (generically
represented as a sequence of 10 Ns). The polymerase is complexed to
a target nucleic acid and a labeled CTP analog. The label on the
CTP analog includes a nucleic acid region having inosines (I) and a
specificity region (A'B'C') that hybridizes to a complementary
region on the tether (ABC).
FIG. 12 shows a tethered polymerase in four different positional
states relative to the charge sensor due to the binding of four
different nucleotide analogs. Each of the nucleotide analogs has an
oligonucleotide moiety of the same length as the other 3 nucleotide
analogs, but each nucleotide analog has a specific binding sequence
that binds to a different region of the tether compared to the
regions where the other nucleotide analogs bind.
DETAILED DESCRIPTION
Embodiments of the present disclosure relate generally to
apparatus, compositions and methods for single molecule detection
useful in applications such as nucleotide incorporation events
detected in nucleic acid sequencing procedures. There is a need for
improved detection systems which provide long sequencing reads in
high-throughput manner. Embodiments of the invention set forth
herein satisfy this need and provide other advantages as well.
Complementary metal-oxide-semiconductor (CMOS)-based sensing
schemes have been used for nucleic acid sequencing. Current
CMOS-based sensing schemes exploit electrochemical detection of the
by-products of the DNA polymerization that occurs in an SBS
reaction (e.g. either protons or pyrophosphate). These methods
provide advantages of being lightless and label-free. Being
lightless, the reactions do not require expensive optics for
detection. Cost and complexity of preparing reagents is typically
reduced when label free reagents are used. However, disadvantages
of current CMOS-based sensing schemes are that the reaction
by-products, that are to be detected, are mobile and have a natural
tendency to diffuse away from the reaction zone, which can result
in cross-talk between neighboring sites when attempting to perform
multiplexed sequencing reactions. Given the large size of most
genomes and the limited read length for typical sequencing
reactions, multiplexing is very important to achieve desired
coverage levels for research and clinical applications of
sequencing technology.
The present disclosure provides a unique detection modality that
can be used for nucleic acid sequencing and for detection of
nucleic acids and other analytes in general. An exemplary
embodiment is shown in FIG. 1. Briefly, polymerase 1 is immobilized
on the gate 5 of a silicon nanowire field-effect transistor (FET) 2
with a tether 3. Optionally, the nanowire can be made of material
other than silicon or the nanowire can be replaced with a nanotube.
Optionally, the tether 3 can be a conductive polymer strand, as
indicated by the positive charge 6 at the end of the tether that is
proximal to the polymerase and the negative charge 7 at the end of
the tether that is distal to the polymerase and attached to the
gate 5. The ssDNA 4 to be sequenced is bound to polymerase 1 after
having been introduced in solution along with nucleotides and other
reactants. As the complimentary strand is synthesized, disturbances
in the charge distribution in the vicinity of the FET 2 are
generated, either as a result of conformational changes of the
polymerase 1, or due to presence of the nucleotides, possibly
modified with an electrically active tag in the vicinity of the FET
2. Those modifications in the charge distribution are sensed by the
nanowire-FET 2 and detected as a modulation in the FET
transconductance current.
Some advantages of the FET-based apparatus and methods set forth
herein are: (1) single-molecule sensitivity can be achieved with a
properly scaled FET (2) high degree of parallelization (also called
"multiplexability") is facilitated since the detected charge
disturbance is localized in the vicinity of the polymerase, thereby
avoiding cross-talk between neighboring FET sites (3) the optional
use of a conducting tether assists in transmitting the charge
disturbance to the gate and minimizes the undesirable effects of
screening from the biological solution and (4) silicon nanowire FET
can be conveniently manufactured using processes that are
compatible with semiconductor manufacturing facilities.
Terms used herein will be understood to take on their ordinary
meaning unless specified otherwise. Examples of several terms used
herein and their definitions are set forth below.
As used herein, the term "array" refers to a population of charge
sensors or molecules that are attached to one or more solid-phase
substrates such that the charge sensors or molecules can be
differentiated from each other according to their relative
location. An array can include different molecules that are each
located at a different addressable location (e.g. at different
charge sensors) on a solid-phase substrate. Alternatively, an array
can include separate solid-phase substrates each bearing a
different molecule, wherein the different probe molecules can be
identified according to the locations of the solid-phase substrates
on a surface to which the solid-phase substrates are attached or
according to the locations of the solid-phase substrates in a
liquid such as a fluid stream. The molecules of the array can be
nucleic acid primers, nucleic acid probes, nucleic acid templates
or nucleic acid enzymes such as polymerases and exonucleases.
As used herein, the term "attached" refers to the state of two
things being joined, fastened, adhered, connected or bound to each
other. For example, a reaction component, such as a polymerase, can
be attached to a solid phase component, such as a charge sensor, by
a covalent or non-covalent bond. A covalent bond is characterized
by the sharing of pairs of electrons between atoms. A non-covalent
bond is a chemical bond that does not involve the sharing of pairs
of electrons and can include, for example, hydrogen bonds, ionic
bonds, van der Waals forces, hydrophilic interactions and
hydrophobic interactions.
As used herein, the term "charge sensor" is intended to mean a
detection device that translates perturbations at its surface or in
its surrounding electrical field into an electrical signal. For
example, a charge sensor can translate the arrival or departure of
a reaction component into an electrical signal. A charge sensor can
also translate interactions between two reaction components, or
conformational changes in a single reaction component, into an
electrical signal. An exemplary charge sensor is a field effect
transistor (FET) such as a carbon nanotube (CNT), single-walled
carbon nanotube (SWNT) based FET, silicon nanowire (SiNW) FET,
graphene nanoribbon FET (and related nanoribbon FETs fabricated
from 2D materials such as MoS2, silicene, etc.), tunnel FET (TFET),
and steep subthreshold slope devices (see, for example, Swaminathan
et al., Proceedings of the 51st Annual Design Automation Conference
on Design Automation Conference, pg 1-6, ISBN: 978-1-4503-2730-5
(2014) and Ionescu et al., Nature 479, 329-337 (2011)). Examples of
FET and SWNT sensors that can be used in the methods and apparatus
of the present disclosure are set forth in US Pat. App. Pub. No.
2013/0078622 A1, which is incorporated herein by reference in its
entirety.
As used herein, the term "cleavable tether" is intended to mean a
chemical linker having a bond that can be selectively broken by a
chemical or physical treatment. Generally, the bond breakage is
selective with respect to the chemical or physical treatment not
having a substantial adverse effect on other reaction components
that are present. A cleavable tether can be susceptible to
selective bond breakage with agents such as, but not limited to,
light, base, acid, heat, enzymes and chemical reagents. Electric
field and physical agitation can also be used to cleave the tether.
In a preferred embodiment the linker is a nucleotide linker. In
some cases the linker comprises a site for cleavage by a sequence
specific restriction endonuclease. However, sequence specific
cleavage sites need not be present in some linkers used in
accordance with methods set forth herein.
As used herein, the term "concatameric repeat" is intended to mean
a serially repeating string of a particular nucleotide sequence in
a single nucleic acid molecule. Concatameric repeats can be
produced, for example, by rolling circle amplification (RCA)
whereby the repeated nucleotide sequence is the complement of the
template that is replicated by RCA.
As used herein, the term "conducting tether" is intended to mean a
chemical linker through which electricity can be conducted or
through which the electrical effects of an electric field can be
transmitted. A conducting tether can be used to chemically link a
reaction component to a charge sensor and to conduct electricity
between the reaction component and the charge sensor. Exemplary
conducting tethers include, but are not limited to, those having a
doped polythiophene, poly(3,4-ethylenedioxythiophene),
polyacetylene, polypyrrole, polyaniline, polyfluorene,
polyphenylene, polypyrene, polyazulene, polynaphthalenes,
polycarbazole, polyindole, or polyazepine structure. Linkers that
can be useful as conducting tethers are also set forth in US Pat.
App. Pub. No. 2010/0073847 A1, which is incorporated herein by
reference in its entirety.
As used herein, the term "conformational signal change" means the
appearance, disappearance, or alteration of a detectable signal
from a molecule in response to a change in the structure, shape or
arrangement of parts of the molecule. For example, the signal
change can be due to a change in the interaction of a label with a
first portion of the molecule to interact with a second portion of
the molecule. The term, when specifically recited, is intended to
distinguish from changes in signal that arise from a label of a
molecule due to a change in the interaction of the label with a
reactant that binds specifically to the molecule or a change in the
interaction of the label with a product that results from catalytic
activity of the molecule.
As used herein, the term "conformationally labeled," when used in
reference to a molecule, means having at least one label that is
responsive to a change in the structure of the molecule, a change
in the shape of the molecule or a change in the arrangement of
parts of the molecule. The molecule can be, for example, a
polymerase, reverse transcriptase, exonuclease or other nucleic
acid enzyme such as those set forth herein below. The parts of the
molecule can be, for example, atoms that change relative location
due to rotation about one or more chemical bonds occurring in the
molecular structure between the atoms. The parts of the molecule
can be domains of a macromolecule such as those commonly known in
the relevant art. For example, polymerases include domains referred
to as the finger, palm and thumb domains. In the case of proteins
the parts can be regions of secondary, tertiary or quaternary
structure. The label(s) can be attached to the molecule, for
example, via a covalent linkage. However, the label(s) need not be
attached to the molecule, being, for example, located in proximity
to the molecule. In particular embodiments the label is not
attached to a reactant or product of the molecule such as a
nucleotide or nucleic acid.
As used herein, the term "different", when used in reference to
nucleic acids, means that the nucleic acids have nucleotide
sequences that are not the same as each other. Two or more
different nucleic acids can have nucleotide sequences that are
different along their entire length. Alternatively, two or more
different nucleic acids can have nucleotide sequences that are
different along a substantial portion of their length. For example,
two or more different nucleic acids can have target nucleotide
sequence portions that are different for the two or more molecules
while also having a universal sequence portion that is the same on
the two or more molecules. The term "different" can be similarly
applied to other molecules, such as polymerases and nucleic acid
enzymes.
As used herein, the term "each," when used in reference to a
collection of items, is intended to identify an individual item in
the collection but does not necessarily refer to every item in the
collection. Exceptions can occur if explicit disclosure or context
clearly dictates otherwise.
As used herein, the term "fluidic communication," when used in
reference to a molecule in a fluid and a site in contact with the
fluid, refers to the ability of the molecule to move in or through
the fluid to contact or enter the site. The term can also refer to
the ability of the molecule to separate from or exit the site to
enter the solution. Fluidic communication can occur when there are
no barriers that prevent the molecule from entering the site,
contacting the site, separating from the site and/or exiting the
site. However, fluidic communication is understood to exist even if
diffusion is retarded, reduced or altered so long as access is not
absolutely prevented.
As used herein, the term "label," when used in reference to a
reaction component, is intended to mean a detectable reaction
component or detectable moiety of a reaction component. A useful
label is a charge label (also called a charge tag) that can be
detected by a charge sensor. A label can be intrinsic to a reaction
component that is to be detected (e.g. a charged amino acid of a
polymerase) or the label can be extrinsic to the reaction component
(e.g. a non-naturally occurring modification of an amino acid). In
some embodiments a label can include multiple moieties having
separate functions. For example a label can include a linker
component (such as a nucleic acid) and a charge tag component.
As used herein, the term "non-natural," when used in reference to a
moiety of a molecule, is intended to refer to a moiety that is not
found attached to the molecule in its natural milieu or in a
biological system unperturbed by human, technical intervention.
Typically, non-natural moieties are synthetic modifications of
molecules that render the molecules structurally or chemically
distinct from the unmodified molecule or from molecules having
natural modifications. As used herein, the term "non-natural," when
used in reference to an analog used for a process, is intended to
mean an analog that is not found in the natural milieu where the
process occurs. Typically, non-natural analogs are synthetic
analogs that are structurally or chemically distinct from other
types of molecules in the class to which the analog belongs.
As used herein, the term "nucleic acid" is intended to be
consistent with its use in the art and includes naturally occurring
nucleic acids or functional analogs thereof. Particularly useful
functional analogs are capable of hybridizing to a nucleic acid in
a sequence specific fashion or capable of being used as a template
for replication of a particular nucleotide sequence. Naturally
occurring nucleic acids generally have a backbone containing
phosphodiester bonds. An analog structure can have an alternate
backbone linkage including any of a variety of those known in the
art such as peptide nucleic acid (PNA) or locked nucleic acid
(LNA). Naturally occurring nucleic acids generally have a
deoxyribose sugar (e.g. found in deoxyribonucleic acid (DNA)) or a
ribose sugar (e.g. found in ribonucleic acid (RNA)).
A nucleic acid can contain any of a variety of analogs of these
sugar moieties that are known in the art. A nucleic acid can
include native or non-native bases. In this regard, a native
deoxyribonucleic acid can have one or more bases selected from the
group consisting of adenine, thymine, cytosine or guanine and a
ribonucleic acid can have one or more bases selected from the group
consisting of uracil, adenine, cytosine or guanine Useful
non-native bases that can be included in a nucleic acid are known
in the art.
As used herein, the term "nucleotide" is intended to include
natural nucleotides, analogs thereof, ribonucleotides,
deoxyribonucleotides, dideoxyribonucleotides and other molecules
known as nucleotides. The term can be used to refer to a monomeric
unit that is present in a polymer, for example to identify a
subunit present in a DNA or RNA strand. The term can also be used
to refer to a molecule that is not necessarily present in a
polymer, for example, a molecule that is capable of being
incorporated into a polynucleotide in a template dependent manner
by a polymerase. The term can refer to a nucleoside unit having,
for example, 0, 1, 2, 3 or more phosphates on the 5' carbon. For
example, tetraphosphate nucleotides and pentaphosphate nucleotides
can be particularly useful. Exemplary natural nucleotides include,
without limitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP,
UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP,
dAMP, dTMP, dCMP, and dGMP.
Non-natural nucleotides also referred to herein as nucleotide
analogs, include those that are not present in a natural biological
system or not substantially incorporated into polynucleotides by a
polymerase in its natural milieu, for example, in a non-recombinant
cell that expresses the polymerase. Particularly useful non-natural
nucleotides include those that are incorporated into a
polynucleotide strand by a polymerase at a rate that is
substantially faster or slower than the rate at which another
nucleotide, such as a natural nucleotide that base-pairs with the
same Watson-Crick complementary base, is incorporated into the
strand by the polymerase. For example, a non-natural nucleotide may
be incorporated at a rate that is at least 2 fold different, 5 fold
different, 10 fold different, 25 fold different, 50 fold different,
100 fold different, 1000 fold different, 10000 fold different or
more when compared to the incorporation rate of a natural
nucleotide. A non-natural nucleotide can be capable of being
further extended after being incorporated into a polynucleotide.
Examples include, nucleotide analogs having a 3' hydroxyl or
nucleotide analogs having a reversible terminator moiety at the 3'
position that can be removed to allow further extension of a
polynucleotide that has incorporated the nucleotide analog.
Examples of reversible terminator moieties that can be used are
described, for example, in U.S. Pat. Nos. 7,427,673; 7,414,116; and
7,057,026 and PCT publications WO 91/06678 and WO 07/123744, each
of which is incorporated herein by reference in its entirety. It
will be understood that in some embodiments a nucleotide analog
having a 3' terminator moiety or lacking a 3' hydroxyl (such as a
dideoxynucleotide analog) can be used under conditions where the
polynucleotide that has incorporated the nucleotide analog is not
further extended. In some embodiments, the nucleotide(s) will not
include a reversible terminator moiety, or the nucleotides(s) will
not include a non-reversible terminator moiety or the nucleotide(s)
will not include any terminator moiety at all. Nucleotide analogs
with modifications at the 5' position are also useful.
As used herein, the term "protection moiety" is intended to mean a
compound or portion thereof that is attached to a reaction
component to prevent the reaction component from undergoing a
particular reaction. For example, a nucleic acid molecule can be
bound to a nucleic acid enzyme such that the nucleic acid molecule
prevents the nucleic acid enzyme from degradation or modification
by a treatment that would otherwise cause degradation or
modification of the enzyme. An antibody can also serve to bind a
reaction component to protect the reaction component from
degradation, inactivation or other reaction.
As used herein, the term "reaction component" is intended to mean a
molecule that takes part in a reaction. Examples include, reactants
that are consumed in a reaction, products that are created by a
reaction, catalysts such as enzymes, that facilitate a reaction,
solvents, salts, buffers and other molecules.
As used herein, the term "repellant moiety" is intended to mean a
molecule or portion thereof that will occupy a space to prevent or
inhibit occupancy of another molecule at the space or to inhibit
juxtaposition of another molecule near the space. A repellant
moiety can act via steric exclusion, charge repulsion,
hydrophobic-hydrophilic repulsion or other forces.
As used herein, the term "terminator moiety," when used in
reference to a nucleotide, means a part of the nucleotide that
inhibits or prevents the nucleotide from forming a covalent linkage
to a second nucleotide. For example, in the case of nucleotides
having a pentose moiety, a terminator moiety can prevent formation
of a phosphodiester bond between the 3' oxygen of the nucleotide
and the 5' phosphate of the second nucleotide. The terminator
moiety can be part of a nucleotide that is a monomer unit present
in a nucleic acid polymer or the terminator moiety can be a part of
a free nucleotide (e.g. a nucleotide triphosphate). The terminator
moiety that is part of a nucleotide can be reversible, such that
the terminator moiety can be modified to render the nucleotide
capable of forming a covalent linkage to a second nucleotide. In
particular embodiments, a terminator moiety, such as a reversible
terminator moiety, can be attached to the 3' position or 2'
position of a pentose moiety of a nucleotide analog.
As used herein, the term "solid support" refers to a rigid
substrate that is insoluble in aqueous liquid. The substrate can be
non-porous or porous. The substrate can optionally be capable of
taking up a liquid (e.g. due to porosity) but will typically be
sufficiently rigid that the substrate does not swell substantially
when taking up the liquid and does not contract substantially when
the liquid is removed by drying. A nonporous solid support is
generally impermeable to liquids or gases. Exemplary solid supports
include, but are not limited to, glass and modified or
functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, Teflon.TM., cyclic
olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica
or silica-based materials including silicon and modified silicon,
carbon, metals, inorganic glasses, optical fiber bundles, and
polymers. Particularly useful solid supports for some embodiments
are located within a flow cell apparatus. Exemplary flow cells are
set forth in further detail below.
As used herein, the term "type" (or "species") is used to identify
molecules that share the same chemical structure. For example, a
mixture of nucleotides can include several dCTP molecules. The dCTP
molecules will be understood to be the same type or species as each
other. Similarly, individual DNA molecules that have the same
sequence of nucleotides are the same type or species.
The embodiments set forth below and recited in the claims can be
understood in view of the above definitions.
The present disclosure provides apparatus, compositions and methods
useful for single molecule detection in applications such as
nucleotide incorporation events detected in nucleic acid sequencing
procedures. The apparatus, compositions and methods set forth
herein are particularly useful, for example, in single molecule
nucleic acid sequencing reactions, such as sequencing by synthesis.
However, it will be appreciated that the apparatus, compositions
and methods set forth herein can be used for any other suitable
detection schemes, including, but not limited to single molecule
detection.
The present disclosure provides a first method of nucleic acid
sequencing. The method can include the steps of (a) providing a
polymerase tethered to a solid support charge sensor; (b) providing
one or more labeled nucleotides, whereby the presence of the label
can be detected by the charge sensor when the label is in proximity
to the charge sensor; and (c) detecting incorporation of the
labeled nucleotide into a nascent strand complementary to a
template nucleic acid.
The polymerase used in the first method of nucleic acid sequencing
can be tethered to the solid support charge sensor with a tether
comprising nucleic acid. For example, the tether can comprise
deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or protein
nucleic acid (PNA).
In some embodiments of the first method of nucleic acid sequencing,
the labeled nucleotide is labeled at the .gamma.-phosphate position
of the nucleotide. The label can comprise an oligonucleotide and,
optionally, the oligonucleotide is capable of hybridizing to an
immobilized nucleic acid.
Further optionally, the immobilized nucleic acid used in the first
method of nucleic acid sequencing is part of a tether, the tether
immobilizing a polymerase to the solid support charge sensor.
In some configurations of the first method of nucleic acid
sequencing, the polymerase is held in proximity of less than 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 nm to the charge sensor.
In some embodiments of the first method of nucleic acid sequencing,
the label is cleaved from the nucleotide after incorporation, for
example, by the polymerase.
Optionally in the first method of nucleic acid sequencing, the one
or more labeled nucleotides comprise a plurality of charge tags.
For example, the one or more labeled nucleotides can comprise a
unique charge tag for each of four types of nucleotides. The charge
tag can be a negative charge tag, for example, comprising one or
more of: a phosphate group, DMT and/or FMOC. Alternatively, the
charge tag can be a positive charge tag, optionally comprising a
primary amine.
The one or more labeled nucleotides in the first method of nucleic
acid sequencing can comprise a plurality of different
oligonucleotides (i.e. oligonucleotide moieties) capable of
hybridizing to a plurality of immobilized tether sequences.
Alternatively or additionally, a plurality of different
oligonucleotides can be capable of hybridizing to a plurality of
different locations within a particular tether used to immobilize a
polymerase.
In some embodiments of the first method of nucleic acid sequencing,
the one or more labeled nucleotides comprise 4 different charge
tags and each of said labeled nucleotides comprises an
oligonucleotide capable of hybridizing to the same immobilized
tether sequence.
In some embodiments of the first method of nucleic acid sequencing,
the polymerase tether and the labels on the labeled nucleotides
contain deoxyribonucleotides that bind complementarily to each
other, ribonucleotides that bind complementarily to each other, or
deoxyribonucleotides that bind complementarily to ribonucleotides.
For example, the polymerase tether and the labels on the labeled
nucleotides can form a DNA:DNA duplex, RNA:RNA duplex or DNA:RNA
heteroduplex. The deoxyribonucleotides or ribonucleotides can be
nucleotide analogs that increase or modify duplex stability. For
example, 2'-O-Methyl (2'-O-Me) or 2'-Fluoro (2'-F) modified
ribonucleotides can be used. The 2'-O-me and 2'-F RNA modifications
are known to increase the melting temperature of RNA:RNA duplexes
by 0.5.degree. C. to 1.degree. C. per base pair, but result in only
small changes in RNA:DNA stability (Majlessi et al., Nucleic Acids
Res. 26:2224-9 (1998), incorporated herein by reference in its
entirety). The increase in stability of the modified RNA:RNA base
pairs can be used to accurately position the tag along the RNA
tether as discussed below.
Moreover, the charge sensor can be functionalized with capture
oligonucleotides and the labeled nucleotide can comprise an
oligonucleotide label which hybridizes to one or more of the
capture oligonucleotides in the first method of nucleic acid
sequencing.
The charge sensor used in the first method of nucleic acid
sequencing can comprise a nanowire FET. Optionally, the charge
sensor comprises a carbon nanotube.
The charge sensor in the first method of nucleic acid sequencing
can be part of an array of charge sensors.
The detecting step of the first method of nucleic acid sequencing
can comprise detecting a plurality of incorporation events in
succession.
The methods and apparatus set forth herein can provide long nucleic
acid sequencing reads; fast reads; high throughput capability for
sequencing; and a scalable platform for sequencing. In some
embodiments, any compromises in single read accuracy can be
mitigated by performing multiple overlapping reads due to the
ability of the methods and apparatus set forth herein to provide
throughput in the number of reads performed in parallel.
An exemplary sensor is shown in FIG. 1. Here a polymerase 1 creates
a reaction site where nucleotides can be incorporated into a primed
DNA template 4. The polymerase 1 is attached to a nanowire FET 2
via a tether 3. The apparatus provides single molecule sensitivity.
Changes in charge distribution at the reaction site (e.g.
polymerase conformation changes, nucleotide incorporation, arrival
or departure of charged tags, changes in proximity of the
polymerase to the charge sensor etc.) transmit to the gate and can
be detected.
In particular embodiments, an apparatus or method of the present
disclosure uses deeply scaled FinFET transistors as single-molecule
charge sensors. FinFet sensors benefit from technology already
under development by leading edge semiconductor manufacturers.
Furthermore, previously published components can be used, including
but not limited to (1) those used for immobilization of lysozyme on
CNT to observe enzyme processivity in real time as described in
Choi et al, Science, 335, 319 (2012), (2) those used to immobilize
the Pol 1 Klenow fragment on CNT and observe DNA processivity in
real time as described in Olsen et al, J. Amer. Chem. Soc., 135,
7885 (2013), (3) those used to elucidate a transduction mechanism
as moving charged residues due to protein allosteric motion as
described in Chi et al, NanoLett 13, 625 (2013). The present
methods can also employ the apparatus, components of the apparatus,
and methods set forth in US Pat. App. Pub. No. 2013/0078622 A1.
Each of the above references is incorporated herein by reference in
its entirety.
The apparatus and methods set forth in US Pat. App. Pub. No.
2013/0078622 A1 provide a Debye screening length of 1-2 nm in
50-100 mM NaCl. In this apparatus the allosteric motion must be
near the attachment point of the polymerase to the transistor.
Also, allosteric motion must be base-dependent to enable real-time
discrimination of different types of nucleotides. Such resolution
has not been previously demonstrated.
An embodiment that can be used to overcome limitations of some
apparatus that utilize allosteric-based detection is diagrammed in
FIG. 2. Here polymerase can be immobilized to a charge sensor such
as a single walled carbon nanotube, silicon nanowire or FinFET.
Immobilization can be via tethers that include DNA, RNA, PNA or
analogs thereof. For convenience of demonstration the diagram shows
four polymerases tethered to the charge sensor, each polymerase
also being bound to a different gamma-phosphate labeled nucleotide
type. As shown, nucleotides have an oligonucleotide moiety attached
to the gamma-phosphate. A beta- or gamma-phosphate-labeled
nucleotide that is properly matched to a template strand of a
target nucleic acid will be held in place by a polymerase that is
also bound to the template long enough to temporarily hybridize the
oligonucleotide moiety to the tether (e.g. via Watson-Crick base
complementarity). The hybridization causes the oligonucleotide
moiety to perturb the field around the charge sensor which produces
a detectable signal due to the change in transistor current through
the charge sensor. The diagram shows the oligonucleotide moiety
entering a field that is within 1-2 nm of the charge sensor. The
properly matched beta- or gamma-phosphate-labeled nucleotide will
be incorporated into a nascent strand that is hybridized to the
template nucleic acid. This will, in turn, break the bond between
the beta phosphate and the newly incorporated nucleotide. As a
result, the oligonucleotide moiety (whether attached at the beta-
or gamma-position of the nucleotide) is free to dehybridize from
the tether and diffuse away from the charge sensor, thereby
returning the field around the sensor to its unperturbed state. The
appearance and disappearance of signal as the field around the
charge sensor is perturbed and returned to the unperturbed state,
respectively, can be correlated with incorporation of a nucleotide
into the nascent strand of the target nucleic acid.
Particular embodiments can exploit synergistic binding of the
gamma-phosphate labeled nucleotide to the polymerase and to the
tether. The stability of the oligonucleotide moiety:tether complex
can be relatively low such that the complex does not form for
gamma-phosphate labeled nucleotide that are not also bound to
polymerase (i.e. gamma-phosphate labeled nucleotides that are free
in solution do not substantially bind to the tether). However, the
synergistic effect of the affinities of the nucleotide moiety for
the polymerase and the oligonucleotide moiety for the tether add up
to allow substantial binding affinity overall. In some embodiments,
the synergistic effect can exploit a combination of specific
binding affinity between the nucleotide label and tether along with
weak affinity produced by non-specific binding interactions. For
example, specific binding can result from standard Watson-Crick
base pairing and non-specific binding interactions can result from
interactions of promiscuous bases (e.g. inosine) with native
nucleotides. Thus, when the gamma-phosphate labeled nucleotide is
bound to polymerase during incorporation, synergistic binding
occurs which greatly increases the stability of the interaction
between oligonucleotide moiety and tether. After the gamma
phosphate is cleaved by the polymerase, the synergistic effect is
lost and the oligonucleotide moiety will dissociate from the
tether.
The type of nucleotide that is incorporated into the nascent strand
at each position of the template strand can be determined based on
unique properties of labels incorporated into each type of
nucleotide. For example, four types of dNTPs can be distinguished
by the position where the oligonucleotide moiety hybridizes to the
tether, the length of the oligonucleotide moiety and/or the
presence of a charged moiety on the label. FIG. 2 provides an
example where four-state discrimination is achieved using 2 charge
tags (other than the negatively charged phosphates of the
oligonucleotide moiety) and two tether hybridization positions.
Specifically, dCTP is uniquely labeled with a negatively charged
extrinsic moiety, dTTP is uniquely labeled with a positively
charged extrinsic moiety, dATP and dGTP are distinguished from the
other two nucleotide types based on absence of any extrinsic charge
moiety, and dATP is distinguished from dGTP based on differential
proximity of the oligonucleotide moieties to the charge sensor when
they are hybridized to the tether.
It will be understood that different nucleotide types can be
distinguished based on any of a variety of combinations of positive
charge moieties, negative charge moieties and/or tether
hybridization locations. Alternatively or additionally, the charge
moieties used to distinguish different types of nucleotides can
differ in the strengths of the charges, even if the charges have
the same sign. The exemplary configuration shown in FIG. 3 provides
four-state discrimination based on a single tether hybridization
position and four different charge moieties. Specifically, dGTP and
dCTP both contain negatively charged moieties that distinguish them
from dATP and dTTP, and dGTP can be distinguished from dCTP due to
charge that is distinguishably higher than the charge on dCTP.
Similarly, dATP and dTTP can be distinguished from each other due
to the higher positive charge on the dATP moiety compared to the
dTTP moiety.
As noted previously herein, the precision of tag placement at
specific hybridization positions along a tether can be enhanced
through the use of a tether having ribonucleotides and a nucleotide
label having 2'-O-Me and 2'F modified RNA bases. Alternative
configurations can use a tether that contains 2'-O-Me and 2'F
modified ribonucleotides with label having ribonucleotides, or both
the tether and label can include a mixture of native
ribonucleotides and 2'-O-Me and 2'F modified ribonucleotides.
Although it is possible to use a tether and/or oligonucleotide
moiety that is primarily composed of RNA, it may be desirable to
use a DNA-based or PNA-based tether and/or oligonucleotide to avoid
nuclease sensitivity that is associated with RNA. For example, a
DNA-based or PNA-based tether and/or oligonucleotide can include
native ribonucleotides or non-native ribonucleotide analogs to
achieve binding advantages set forth herein while reducing risk of
unwanted nuclease digestion. In further embodiments, the tether can
include one or more deoxyribonucleotides that are complementary to
ribonucleotides in a nucleotide label or alternatively the tether
can include ribonucleotides that are complementary to
deoxyribonucleotides in a nucleotide label.
A tether that attaches a polymerase to a charge sensor can have
different binding positions for different nucleotide analogs as set
forth in several exemplary embodiments herein. The binding
positions for two or more nucleotide analogs can overlap or they
can be discrete with no overlap. For example, as shown in FIG. 10,
the binding sites for ATP and GTP analogs overlap on the tether by
2 nucleotides (i.e. there is a 1 nucleotide offset between the two
binding sites). For purposes of illustration, the tether sequence
is depicted as a series of generic "N" nucleotides. Any of a
variety of sequences can be used in accordance with rules of
complementarity and desired hybridization strengths and
specificities. As also shown in FIG. 10, the binding sites for ATP
and TTP on the tether have no overlap, being discrete and separated
by 1 nucleotide. Depending on the length of the tether, length of
the binding sites and length of the oligonucleotide moieties on the
nucleotide analogs, some, all or none of the binding sites on the
tether can overlap.
The oligonucleotide moiety of a nucleotide analog can have a
sequence of nucleotides that hybridizes specifically to a
complementary sequence on a tether. In some embodiments the
oligonucleotide moiety can also include promiscuous nucleotide
positions that bind non-specifically to a tether. Such positions
can provide a weak interaction between the oligonucleotide moiety
and tether that facilitates the formation of a specific hybrid
structure. For example, as shown in FIG. 11, an oligonucleotide
moiety can include several inosines (I) that are known to bind
promiscuously, albeit weakly, with all four native nucleotides of
DNA. The oligonucleotide moiety and tether can form a weak complex
via interactions between the inosines in the oligonucleotide moiety
and the native nucleotides in the tether. This can allow the
specific portions of the sequence (e.g. indicated as ABC and its
complement A'B'C' in the figure) to associate more rapidly than
they would have if required to diffuse absent formation of a weak
complex. Furthermore, once a specific complex has formed the
inosines can provide further stability.
The exemplary oligonucleotide moiety in FIG. 11 includes
promiscuous nucleotide positions flanking both sides of the
specific sequence. However, it will be understood that one or more
promiscuous nucleotide positions can be located on only the 5' or
3' side of the specific sequence. Other examples of promiscuous
nucleotide positions include those formed by degenerate
oligonucleotide synthesis or those formed with other nucleotide
analogs known in the art to hybridize promiscuously with 2 or more
types of nucleotides.
In the examples shown in FIG. 10 and FIG. 11, as well as other
examples set forth herein, the tether is a nucleic acid that
hybridizes to an oligonucleotide moiety of a nucleotide analog. It
will be understood that other binding partners can be used as
tether and label moiety instead of the nucleic acids. The binding
sites can be discrete or overlapping as exemplified above for
nucleic acids. Also, the binding sites can include a combination of
weak, non-specific interacting partners along with stronger,
specific interacting partners.
Several embodiments set forth herein have exemplified the use of a
plurality of different nucleotide analogs having oligonucleotide
moieties of differing lengths. In such embodiments, the different
nucleotide types can be distinguished based on the different
lengths of the oligonucleotide moieties. Alternatively, different
nucleotide analogs can have oligonucleotide moieties of the same
length. However, each nucleotide analog can have a specific binding
sequence that binds to a different region of a tether compared to
the regions where the other nucleotide analogs bind. An exemplary
configuration is shown in FIG. 12 where binding of the polymerase
to different nucleotide analogs places the polymerase in one of
four distinguishable states. The oligonucleotide moiety for the ATP
analog binds to a location on the tether that is nearest to the
attachment point of the tether to the polymerase, the
oligonucleotide moiety for the TTP analog binds to a location on
the tether that is furthest from the attachment point of the tether
to the polymerase, and the oligonucleotide moieties for the GTP and
CTP analogs bind to respectively distinct locations on the tether
that are at intermediate distances from the binding sites for the
other two nucleotide analogs. As such, binding of the different
nucleotide analogs to the polymerase will position the polymerase
at different distances from the charge sensor (e.g. causing
different size loops to form in the tether as shown in the figure).
The different nucleotide types can be distinguished based on the
differences in signals produced for the different distances of the
polymerase from the sensor. In embodiments where one or more of the
nucleotide analogs includes a charge tag or other detectable moiety
(e.g. attached at the end of the oligonucleotide moiety that is
distal to the nucleotide moiety), the binding between the
oligonucleotide moiety and tether will position the detectable
moiety at different distances from the charge sensor. In this case,
the different nucleotide types can be distinguished based on the
differences in signals produced for the different distances of the
detectable moieties from the sensor.
As demonstrated by the embodiment diagrammed in FIG. 4, the tether
that attaches polymerase to the charge sensor need not be capable
of hybridizing to the tags present on the nucleotides. Rather, the
charge sensor can be functionalized to attach one or more
oligonucleotides that are complementary to one or more of the
nucleotide types being detected. Discrimination of the different
nucleotides can be achieved based on sign of the charge, strength
of the charge, length of the oligonucleotide moiety that hybridizes
to the surface attached oligonucleotide(s), or proximity/location
on the surface attached oligonucleotide(s) where the
oligonucleotide moiety hybridizes, or a combination thereof.
Advantages of several configurations set forth above include, for
example, overcoming screening issues by placing charges within 1-2
nm of the gate with atomic precision, ability to achieve a higher
level of current modulation through the use of charge tags, and
opening up of the space of available polymerases since
base-specific allosteric motion is not required for detection.
An exemplary charge tag that can be useful in the apparatus and
methods set forth herein is a phosphate moiety, for example,
located at the 5' end of a nucleic acid moiety. This moiety can be
readily added during available oligonucleotide synthesis protocols
and will result in two negatively charged oxygens at the end of the
oligonucleotide moiety as shown in FIG. 5. Chemical phosphorylation
during oligonucleotide synthesis can be achieved by converting a
DMT protecting group into a 5' phosphate group using 2-[2-(4,4'
Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-p-
hosphoramidite (available from Glen Research, Sterling Va., catalog
No. CPR 10-1900). A series of charge tags having different numbers
of negative charges can be made using
Tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoeth-
yl)-(N,N-diisopropyl)]-phosphoramidite (available from Glen
Research, Sterling Va., catalog No. 10-1922-xx),
1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)--
(N,N-diisopropyl)]-phosphoramidite (available from Glen Research,
Sterling Va., catalog No. 10-1920-xx),
1-[5-(4,4'-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonylox-
ypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
(available from Glen Research, Sterling Va., catalog No.
10-1921-xx), or oligonucleotide dendrimers which contain various
numbers of DMT (4,4'-dimethoxytrityl) or Fmoc
(Fluorenylmethyloxycarbonyl) moieties, such as those available from
Glen Research or shown in FIG. 6. A useful positively charged tag
is
2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropy-
l)-phosphoramidite (available from Glen Research, Sterling Va.,
catalog No. 10-1905-xx). Another useful positively charged moiety
is a 5' primary amine which would have a single positive charge at
the appropriate pH.
Table I provides a listing of useful modifications and charges that
can be used as labels in an apparatus or method set forth
herein.
TABLE-US-00001 TABLE I 5' Terminus Reagents Final Charge State 5'
OH N/A Neutral 5' Phosphate CPR 10-1900 (Glen Res.) -2 5' Phosphate
(x2) CPR 10-1900 and symmetric -4 doubler (Glen Res.) 5' Phosphate
(x3) CPR 10-1900 and symmetric -6 trebler (Glen Res.) 5' primary
amine 5' amino-modifier 5 +1
The present disclosure provides a method for attaching reaction
components to charge sensors. The method can include the steps of
(a) providing a solid support including a plurality of charge
sensors, wherein each of the charge sensors has a capacity to
attach a plurality of reaction components; (b) providing a fluid
containing a plurality of reaction components of a particular type;
and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; and (iii) reaction components of the
particular type from the fluid attach to the charge sensors under
conditions that result in a solid support where each of the charge
sensors is attached to a single one of the reaction components.
In some embodiments the method for attaching reaction components to
charge sensors can include the steps of (a) providing a solid
support including a plurality of charge sensors, wherein each of
the charge sensors has a capacity to attach a plurality of reaction
components; (b) providing a fluid containing a plurality of
reaction components of a particular type, wherein each of the
reaction components of the particular type is bound to a repellant
moiety; and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; (iii) reaction components from the fluid
attach to the charge sensors, and (iv) the repellant moiety bound
to each of the reaction components prevents more than one of the
reaction components in the plurality of reaction components from
attaching to each of the charge sensors.
A method for attaching reaction components to charge sensors can
include the steps of (a) providing a solid support including a
plurality of charge sensors, wherein each of the charge sensors has
a capacity to attach a plurality of reaction components; (b)
providing a fluid containing a plurality of reaction components of
a particular type; (c) contacting the solid support with the fluid
under conditions wherein (i) the plurality of reaction components
of the particular type are in fluid communication with the
plurality of charge sensors, (ii) a greater number of reaction
components of the particular type is in the fluid than the number
of charge sensors on the solid support; and (iii) reaction
components of the particular type from the fluid attach to the
charge sensors, thereby forming modified charge sensors that are
attached to multiple reaction components of the particular type
from the fluid; and (d) removing one or more of the reaction
components of the particular type from each of the modified charge
sensors to leave a single one of the reaction components of the
particular type attached to each of the modified charge
sensors.
Useful charge devices include analytical devices that can
incorporate a reaction component in direct spatial contact with a
transduction element in a way to allow the rapid and convenient
conversion of reaction events to detectable signals. Devices based
on field-effect transistors (FETs) can directly translate
interactions between reaction components (e.g., polymerases) and
the transistor surface into readable electrical signals. In a
standard FET, current flows along a conducting path (the channel)
that is connected to two electrodes, (the source and the drain).
The channel conductance between the source and the drain is
switched on and off by a third (gate) electrode that can be
capacitively coupled through a thin dielectric layer.
In particular embodiments, FETs are configured to accomplish single
molecule detection. More particularly, these charge sensors can be
configured to monitor the dynamics of a single molecule reaction.
Any type of conduction channel that is generally found in field
effect transistors can be used in an apparatus or method set forth
herein. Exemplary conduction channels are formed from metals, metal
oxides, semiconductors, or nanometer-scale conductors such as
nanowires, or graphene.
Particularly useful charge sensors for single molecule detection
are single-walled carbon nanotubes (SWNTs). See, for example, Star
et al., Nano. Lett. 3, 459 (2003); Star et al., Org. Lett. 6, 2089
(2004); Besterman et al., Nano. Lett. 3, 727 (2003); Gruner, Anal.
Biooanal. Chem. 384, 322 (2005); Chen et al. Proc. Natl. Acad. Sci.
U.S.A. 100, 4984 (2003) and US Pat App. Pub. No. 2013/0078622 A1,
each of which is incorporated herein by reference in its entirety.
SWNTs are extremely small conductors, typically on the order of
about 1 nanometer in diameter.
A SWNT can be coated with a chemoselective polymer, metal or metal
oxide nanoparticle, or reaction components like proteins, nucleic
acids or antibodies. See for example, Besterman et al., Nano. Lett.
3, 727 (2003); and Chen et al. Proc. Natl. Acad. Sci. U.S.A. 100,
4984 (2003). Single reaction components can be attached to these
SWNT and other charge sensors using methods set forth herein.
In some embodiments a single reaction component can be attached to
a charge sensor by creating one single covalent defect on the
charge sensor, for example, using techniques set forth in Goldsmith
et al. Science 315, 77 (2007), which is incorporated herein by
reference in its entirety. For example a SWNT can be produced
having a single defect such that a variety of attachment
chemistries can be used to link a single reaction component to the
reactive defect site selectively, without coating the rest of the
SWNT with additional reaction components. SWNTs can also be
attached to reaction components by non-covalent means, for example,
using techniques set forth in Chen et al, J. Am. Chem. Soc. 123,
3838 (2001), which is incorporated herein by reference in its
entirety. These methods can be modified as set forth herein to
reliably bind a single reaction component non-covalently to a
SWNT.
SWNTs are semiconductors with electronic bandgaps that can vary
from 1 electron volt to effectively zero. SWNTs are useful as
conduction channels because single molecule sensing devices can be
fabricated from SWNT wires of any type, with or without gate
electrodes, and on glass, plastic, or silicon substrates. Useful
SWNTs and their configurations for single molecule detection are
set forth in US Pat App. Pub. No. 2013/0078622 A1, which is
incorporated herein by reference in its entirety.
Other charge sensors that can be modified for use in an apparatus
or method set forth herein include, without limitation, silicon
nanowire (SiNW) FET, FET made of III-V materials, silicon FinFET,
graphene nanoribbon FETs as well as nanoribbon FETs from other 2D
materials such as MoS2 and silicene, tunnel FET (TFET), and steep
subthreshold slope devices (see, for example, Swaminathan et al.,
Proceedings of the 51st Annual Design Automation Conference on
Design Automation Conference, pg 1-6, ISBN: 978-1-4503-2730-5
(2014) and Ionescu et al., Nature 479, 329-337 (2011)). Carbon
nanotubes can also be useful.
A plurality of charge sensors can be provided in the form of an
array of charge sensors. The array can include at least 10, 100,
1.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.4,
1.times.10.sup.4 or more charge sensors. Each individual charge
sensor can be located at a discrete location in the array that is
separated from the other charge sensors in the array. For example,
each charge sensor can reside in a well or depression in a solid
support. The locations, even when separated from each other, can
optionally be in fluid contact with a bulk solution. In such a
configuration, multiplex reactions can occur on the array of charge
sensors by delivering common reagents to all of the charge sensors
via bulk fluid delivery. Taking nucleic acid sequencing reactions
as an example, nucleotides can be delivered via bulk solution to an
array of wells (or other features), each well (or other feature)
hosting an individual sequencing reaction. The nucleotide delivery
will result in parallel sequencing reactions at the wells (or other
features).
A charge sensor, such as a Si nanowire can have dimensions that are
less than 10 nm wide and greater than 100 nm long. A Si nanowire or
other charge sensor can be placed in a well that is 10 nm.times.10
nm, 50 nm.times.100 nm or larger. For example, a well within which
a charge sensor resides can have an opening on a surface that is at
least 100 nm.sup.2, 1000 nm.sup.2, 5000 nm.sup.2, 1.times.10.sup.4
nm.sup.2, or larger. The circuitry to read out the signal from the
charge sensing element can occupy an area of the solid support that
is 1 micron.times.1 micron or larger.
In some embodiments, a charge sensor will have a uniform width
along the entire length. Alternatively, a charge sensor can have
substantial variable width along its length. For example, a charge
sensor can include a region of relatively narrow width, akin to a
`pinched` region. The width of the pinch region can have a diameter
that is, for example, 75%, 50%, 40%, 30%, 20% or 10% of the
diameter of the relatively larger width regions of the charge
sensor. The pinch can provide the advantage of increasing the
effect of a polymerase's charged residue motion on the sensor. A
further benefit is relaxed tolerances for channel fabrication and
opening/alignment of the first layer dielectric passivation of the
FET sensor. For example a charge sensor having a 20 nm diameter
with a 10 nm pinched area can be particularly useful. Other
dimensions for a charge sensor with a pinched region include, for
example diameters of at least 25, 30, 35, or 40 nm or more with a
pinched region that is at most 10, 20, 30, 40, or 50 nm long.
Sensors having smaller diameters can also be used for example those
having diameters of at least 5, 10, 15, or 20 nm can have a pinched
region with a length in the exemplified range. It will be
understood that in some embodiments the maximum diameter of a
charge sensor will be 50 nm, 40 nm, 30 nm, 20 nm 10 nm or less.
A tether for a polymerase or other enzyme can be attached to a
pinched region or at a point that is proximal to the pinched
region. Thus, the tether can be placed to localize a polymerase or
other enzyme in proximity to the pinched region.
An exemplary implementation of a charge sensor having a pinched
region is shown in FIG. 8. The pinched region can be fabricated via
appropriate lithographic mask design where the shape of the mask is
such that it produces the desired pinch. Alternatively, proximity
effects during lithographic patterning may be used to locally pinch
the charge sensor by placing electrodes orthogonal to the sensor in
close proximity to the region to be pinched. In addition to
producing the desired pinch, the proximity electrodes can be used
to produce an electrophoretic force that positions the enzyme at
the pinch region.
Another exemplary implementation is shown in FIG. 9. Here a pinch
is introduced into silicon using local oxidation. In a first step a
silicon wire is coated with an oxidation barrier, such as Silicon
Nitride (SiNx). A region of the silicon wire can be exposed using
local lithography to etch away the oxidation barrier. Then the
partially coated wire can be oxidized to reduce the width of the
silicon wire at the exposed region. Further etching can be carried
out to expose the silicon pinch for chemical conjugation.
Those skilled in the art will recognize that the above methods for
producing a pinch in a charge sensor are exemplary and not
limiting; a pinch may be produced with a number of other methods
common in semiconductor manufacturing, e.g., through the use of
sacrificial layers, selective deposition and etch, or additional
lithographic masks, among others.
The density of an array can be from 2 to as many as a billion or
more different reaction sites per square cm. Very high density
arrays are useful in the invention including, for example, those
having at least about 10,000,000 reaction sites/cm.sup.2,
including, for example, at least about 100,000,000 reaction
sites/cm.sup.2, 1,000,000,000 reaction sites/cm.sup.2, up to about
2,000,000,000 reaction sites/cm.sup.2 or higher. High density
arrays can also be used including, for example, those in the range
from about 100,000 reaction sites/cm.sup.2 to about 10,000,000
reaction sites/cm.sup.2. Moderate density arrays useful in the
invention can range from about 10,000 reaction sites/cm.sup.2 to
about 100,000 reaction sites/cm.sup.2. Low density arrays are
generally less than about 10,000 reaction sites/cm.sup.2.
Any of a variety of reaction components can be attached to a charge
sensor. For example, a receptor, such as an antibody or lectin; a
ligand, such as a nucleotide, epitope, carbohydrate, or drug
candidate; a nucleic acid such as target nucleic acid, tether
nucleic acid or other nucleic acid set forth in connection with a
reaction set forth herein; or an enzyme, such as a polymerase or
other nucleic acid binding enzyme, a kinase, phosphatase,
exonuclease, protease, or metabolic enzyme. Other useful reaction
components include, but are not limited to, components of reactions
set forth herein or known in the art of molecular biology or
biochemistry.
Multiplex embodiments, including, for example, those that employ an
array of charge sensors can be configured such that a single type
of reaction component is attached to each charge sensor. For
example, the charge sensors in a multiplex embodiment can
substantially all be attached to a polymerase. Furthermore, the
same species of polymerase can be attached to each of the charge
sensor. This configuration can provide an expected uniform output
from each charge sensor, but for differences in the other reaction
components that come into contact with each respective charge
sensor. Such a configuration can be achieved by providing a fluid
that is homogeneous with respect to having a single type of
polymerase when attaching the polymerases to the charge
sensors.
Any of a variety of polymerases can be used in a method or
composition set forth herein including, for example, protein-based
enzymes isolated from biological systems and functional variants
thereof. Reference to a particular polymerase, such as those
exemplified below, will be understood to include functional
variants thereof unless indicated otherwise. A particularly useful
function of a polymerase is to catalyze the polymerization of a
nucleic acid strand using an existing nucleic acid as a template.
Other functions that are useful are described elsewhere herein.
Examples of useful polymerases include DNA polymerases and RNA
polymerases. Exemplary DNA polymerases include those that have been
classified by structural homology into families identified as A, B,
C, D, X, Y, and RT. DNA Polymerases in Family A include, for
example, T7 DNA polymerase, eukaryotic mitochondrial DNA Polymerase
.gamma., E. coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus
stearothermophilus Pol I. DNA Polymerases in Family B include, for
example, eukaryotic DNA polymerases a, 6, and E; DNA polymerase C;
T4 DNA polymerase, Phi29 DNA polymerase, and RB69 bacteriophage DNA
polymerase. Family C includes, for example, the E. coli DNA
Polymerase III alpha subunit. Family D includes, for example,
polymerases derived from the Euryarchaeota subdomain of Archaea.
DNA Polymerases in Family X include, for example, eukaryotic
polymerases Pol .beta., pol .sigma., Pol .lamda., and Pol .mu., and
S. cerevisiae Pol4. DNA Polymerases in Family Y include, for
example, Pol .eta., Pol iota, Pol kappa, E. coli Pol IV (DINB) and
E. coli Pol V (UmuD'2C). The RT (reverse transcriptase) family of
DNA polymerases includes, for example, retrovirus reverse
transcriptases and eukaryotic telomerases. Exemplary RNA
polymerases include, but are not limited to, viral RNA polymerases
such as T7 RNA polymerase; Eukaryotic RNA polymerases such as RNA
polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase
IV, and RNA polymerase V; and Archaea RNA polymerase.
The above classifications are provided for illustrative purposes.
It will be understood that variations in the classification system
are possible. For example, in at least one classification system
Family C polymerases have been categorized as a subcategory of
Family X. Furthermore, polymerases can be classified according to
other characteristics, whether functional or structural, that may
or may not overlap with the structural characteristics exemplified
above. Some exemplary characteristics are set forth in further
detail below.
A polymerase having an intrinsic 3'-5' proofreading exonuclease
activity can be useful for some embodiments. Polymerases that
substantially lack 3'-5' proofreading exonuclease activity are also
useful in some embodiments, for example, in most sequencing
embodiments. Absence of exonuclease activity can be a wild type
characteristic or a characteristic imparted by a variant or
engineered polymerase structure. For example, exo minus Klenow
fragment is a mutated version of Klenow fragment that lacks 3'-5'
proofreading exonuclease activity. Klenow fragment and its exo
minus variant can be useful in a method or composition set forth
herein. On the other hand, the large fragment of A-family DNA
polymerases, such as Bsu DNA polymerase I (Bsu-LF), naturally lack
a 3' to 5' exonuclease function.
Polymerases having 3' to 5' exonuclease activity undergo
intramolecular and intermolecular switching as described, for
example, in Lamichhane et al. J. Am. Chem. Soc. 135:4735-4742
(2013), which is incorporated herein by reference in its entirety.
In some embodiments, it is desirable to use a polymerase that lacks
3' to 5' exonuclease activity. For example, in some embodiments the
switching can cause a conformational change that is difficult to
distinguish from one or more of the conformational changes that
occur due to polymerase activity and that are utilized for
sequencing or other analyses. The 3' to 5' exonuclease activity can
be removed by removing all or part of the 3' to 5' exonuclease
domain or by introducing loss of function mutations into the 3' to
5' exonuclease domain.
The large fragment of A-family DNA polymerases, such as Bsu DNA
polymerase I (Bsu-LF), can be further modified for use in a method
set forth herein. Native Bsu-LF has no cysteine (Cys) residues. One
or more Cys residue can be engineered at a surface accessible
location of Bsu-LF to allow for a convenient attachment site for a
tether or other linker having a sulfhydryl reactive moiety.
Candidate sites for introduction of a Cys mutation can be
determined from inspection of crystal structures of Bsu-LF or other
homologous A-family polymerases.
It may also be desirable to engineer Bsu-LF to introduce charged
residues that will interact favorably with a charge sensor. For
example, a beneficial modification is to introduce one or more
residues from the O-helix finger domain of Klenow fragment into
comparable position(s) of Bsu-LF, the net result of which is to
introduce more charged amino acids into the mutant Bsu-LF. See FIG.
7 for the location of the O-helix finger domain of Klenow fragment
(Pollxxo in the figure) and the location to be replaced in Bsu-LF
(Bsuxxx1) in the figure. Similar changes can be made to other
A-family polymerases such as Taq DNA polymerase or Bst DNA
polymerase
Polymerases can be characterized according to their processivity. A
polymerase can have an average processivity that is at least about
50 nucleotides, 100 nucleotides, 1,000 nucleotides, 10,000
nucleotides, 100,000 nucleotides or more. Alternatively or
additionally, the average processivity for a polymerase used as set
forth herein can be, for example, at most 1 million nucleotides,
100,000 nucleotides, 10,000 nucleotides, 1,000 nucleotides, 100
nucleotides or 50 nucleotides. Polymerases can also be
characterized according to their rate of processivity or nucleotide
incorporation. For example, many native polymerases can incorporate
nucleotides at a rate of at least 1,000 nucleotides per second. In
some embodiments a slower rate may be desired. For example, an
appropriate polymerase and reaction conditions can be used to
achieve an average rate of at most 500 nucleotides per second, 100
nucleotides per second, 10 nucleotides per second, 1 nucleotide per
second, 1 nucleotide per 10 seconds, 1 nucleotide per minute or
slower. As set forth in further detail elsewhere herein, nucleotide
analogs can be used that have slower or faster rates of
incorporation than naturally occurring nucleotides. It will be
understood that polymerases from any of a variety of sources can be
modified to increase or decrease their average processivity or
their average rate of processivity (e.g. average rate of nucleotide
incorporation) or both. Accordingly, a desired reaction rate can be
achieved using appropriate polymerase(s), nucleotide analog(s),
nucleic acid template(s) and other reaction conditions.
Depending on the embodiment that is to be used, a polymerase can be
either thermophilic or heat inactivatable. Thermophilic polymerases
are typically useful for high temperature conditions or in
thermocycling conditions such as those employed for polymerase
chain reaction (PCR) techniques. Examples of thermophilic
polymerases include, but are not limited to 9.degree. N DNA
Polymerase, Taq DNA polymerase, Phusion.RTM. DNA polymerase, Pfu
DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and
VentR.RTM. DNA polymerase. Most polymerases isolated from
non-thermophilic organisms are heat inactivatable. Examples are DNA
polymerases from phage. It will be understood that polymerases from
any of a variety of sources can be modified to increase or decrease
their tolerance to high temperature conditions. A heat spike (i.e.
brief time period of increased temperature) can be used to
inactivate one or more heat inactivatable polymerases in an array
while leaving thermophilic polymerases in an active state for
subsequent reactions or for subsequent cycles of a sequencing
reaction.
In an alternative embodiment, several different types of reaction
component can be attached across the multiplex collection of charge
sensors. For example, a first subset of charge sensors in an array
can be attached to a first species of polymerase and a second
subset of charge sensors in the array can be attached to a second
species of polymerase. Two, three, or more species of polymerase
can be used. The use of different species of polymerase can be
useful when the different polymerases have different specificity or
sensitivity for different types of nucleotides or different
template sequences. For example, the different types of polymerases
can produce mutually distinguishable signals detectable by the
charge sensors when incorporating the same type of nucleotide into
a nascent strand of a nucleic acid. In another example, the
different types of polymerases can include at least one DNA
polymerase and at least one RNA polymerase. Such a configuration
can be achieved by providing a fluid that is heterogeneous with
respect to having multiple type of polymerase when attaching the
polymerases to the charge sensors.
In some configurations, a charge sensor will have a capacity to
attach more than one reaction component of a particular type. For
example, a charge sensor may have a capacity to attach more than
one polymerase. Depending on the size of the charge sensor and
volume occupied by the polymerase, the charge sensor may have a
capacity to attach at least 2, 3, 4, 5, 10, 15, 25 or more
polymerases. This can be the case for a plurality of charge sensors
in an array. As will be set forth in further detail below,
conditions can be temporarily imposed to decrease the capacity of a
charge sensor, increase the steric bulk of a polymerase (or other
reaction component), or otherwise favor attachment of a single
polymerase (or other reaction component) to an individual charge
sensor. Again, the conditions can be applied to an array of charge
sensors.
A reaction component can be attached to a charge sensor using any
of a variety of chemistries known in the art. For example, chemical
linkers can be used. In many embodiments, the surface of the charge
sensor is one of SiO.sub.2, Al.sub.2O.sub.3, HfO.sub.2,
Ta.sub.2O.sub.5. Other oxides can also be used, for example from
the lanthanide group. Nitrides and oxinytrides are also possible.
The attachment to a linker can conveniently be made through a
surface hydroxyl. In particular embodiments, the linker molecule
includes at least a first and a second functional group. Generally,
the first functional group interacts with the charge sensor and the
second functional group interacts with the reaction component.
Exemplary first functional groups include a pyrene, a benzene, a
cyclohexane, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. An
exemplary second functional group is maleimide. Other chemistries
known to covalently link proteins to surfaces or other moieties can
be used such as those sold by Thermo Fisher (Waltham, Mass.), or
Sigma Aldrich (St. Louis, Mo.). The chemical group on the
polymerase attached to the tethers can be thiol, amine or
carboxylic group. In certain embodiments in which the conduction
channel is a SWNT, the surface of a SWNT is a roughly one atom
thick layer of graphite. The linker molecule can be covalently
linked to one or few carbon atom, or it can interact with a
sidewall of the SWNT through pi-pi stacking.
A reaction component can be attached to a charge sensor by a
non-covalent linkage such as one formed between a receptor and a
ligand. Particularly useful linkages are those between streptavidin
(or variants or analogs thereof) and biotin (or its analogs), those
between complementary nucleic acids, those between antibodies and
epitopes and the like. Members of the above pairs can be linked to
a reaction component and charge sensor, respectively, such that
contacting a fluid containing the reaction components with a solid
support having the charge sensor will result in formation of the
noncovalent bond that tethers the reaction component to the charge
sensor.
In some embodiments, the reaction components from the fluid attach
to the charge sensors to form a conducting tether. Exemplary
conducting tethers include those having a structure that includes
doped polythiophene, poly(3,4-ethylenedioxythiophene),
polyacetylenes, polypyrroles, polyanilines, polyfluorenes,
polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes,
polycarbazoles, polyindoles, or polyazepines. Charge doping of
these tether structures can be achieved by oxidation of the
polymer. Exemplary conducting tethers and methods for their
creation are set forth in Vernitskaya et al. Russ. Chem. Rev.
66:443ff (1997); MacDiarmid, Angew. Chem., Int. Ed. 40:2581-2590
(2001); or McNeill et al., Aust. J. Chem. 16:1056-75 (1963), each
of which is incorporated herein by reference in its entirety.
In particular embodiments, a solid support can be within or part of
a vessel such as a well, tube, channel, cuvette, Petri plate,
bottle or the like. A particularly useful vessel is a flow-cell,
for example, as described in US 2010/0111768 A1 or Bentley et al.,
Nature 456:53-59 (2008), each of which is incorporated herein by
reference in its entirety. Exemplary flow-cells are those that are
commercially available from Illumina, Inc. (San Diego, Calif.).
Flow cells are convenient for delivering bulk reagents to an array
of charge sensors during attachment of reaction components to the
charge sensors or during subsequent reactions carried out with the
reaction components on the charge sensors. Cyclic processes such as
nucleic acid sequencing reactions are particularly well suited for
flow cell devices. Another particularly useful vessel is a well in
a multiwell plate or microtiter plate.
A method set forth herein can include steps of contacting a
plurality of charge sensors with a fluid containing a plurality of
reaction components of a particular type under conditions wherein
the reaction components are in fluid communication with the
plurality of charge sensors and the reaction components attach to
the charge sensors. The result can be that each of the charge
sensors becomes attached to a single one of the reaction component
even when number of reaction components in the fluid is greater
than the number of charge sensors that are contacted by the fluid.
The fraction of the charge sensors that attach to one and only one
of the reaction components of the particular type would be expected
to conform to the Poisson distribution. The Poisson distribution
sets a maximum of 37% occupancy for the fraction of charge sensors
that would attach to only a single reaction component of a
particular type when those reaction components were delivered to
the charge sensors in a bulk fluid. However, in accordance with the
methods set forth herein, bulk fluid delivery of reaction
components of a particular type (e.g. polymerase or other nucleic
acid enzyme) can result in greater than 35%, 40%, 50%, 75%, 90%,
95% or 99% of the charge sensors in the plurality being occupied by
a single reaction component of the particular type.
In some embodiments of the methods, reaction components can be
transported from bulk solution to charge sensors, for example, by
diffusion or other passive process. Attachment of the reaction
components to the charge sensors can thus occur, for example, in
accordance with chemistries set forth herein or known in the art.
Alternatively, reaction components can be actively transported to
the charge sensors, for example, via electric field (e-field)
assisted transport. Again, attachment of the reaction components to
the charge sensor can result using chemistries set forth herein or
known in the art.
A method set forth herein can be modified to use electric field
(e-field) assisted transport of reaction components to sites that
contain charge sensors. For example, each charge sensor on a solid
support can be present at a site that is electrically coupled to a
power source to produce an electric charge that attracts
polymerases or other reaction components to that site and into
proximity with the charge sensor at that site. Exemplary methods
and apparatus for using e-field assist to attract analytes to sites
of an array are described in US 2009/0032401 A1, which is
incorporated herein by reference in its entirety. E-field assist
can be used in a method of the present disclosure, for example,
under conditions where a plurality of different polymerases (or
other reaction components) is in solution such that the polymerases
are in fluidic communication with the plurality of charge sensors.
The charge at the site of each charge sensor can be adjusted to
achieve a desired rate or amount of transport for the polymerase
(or any other particular type of reaction component).
In particular embodiments that utilize e-field assisted transport,
the e-field can be consistently applied throughout the course of
the reaction that is used to attach a polymerase (or any other
particular type of reaction component) to a charge sensor.
Alternatively, the e-field can be changed (e.g. increased or
decreased) as the attachment reaction progresses and charge sensors
fill with polymerase (or any other particular type of reaction
component). For example, increasing the e-field can provide the
benefit of increasing the number of charge sensors that attach to a
polymerase. The rate at which the e-field is increased, and the
amplitude range for the increase, can be selected to balance the
increasing rate of reaction component transport to charge sensors
over time with the increasing number of charge sensors that have
become attached to polymerase over that same period of time. The
rate of change for the e-field can be based on a predicted or
expected rate of polymer attachment. Alternatively, the e-field can
be changed in response to empirical detection of polymerase
attachment to the charge sensors as set forth in further detail
herein.
In particular embodiments, an e-field can be applied substantially
uniformly to all of the sites of an array that have charge sensors.
Thus, polymerases (or other reaction components) that are in
solution will have an equal probability of being transported to any
given charge sensor. In an alternative embodiment, an e-field can
be applied to only a subset of the charge sensor sites that are
present in an array. In this way, e-field assist can be used to
selectively attach polymerase (or other reaction component) to some
charge sensors over others. Furthermore, if desired, an attractive
charge can be applied at a first subset of charge sensor sites in
order to transport polymerase to the first subset of sites and in
the meantime a repellant charge can be applied to a second subset
of charge sensor sites to inhibit polymerases from being
transported to those sites or to remove (e.g. via desorption or
degradation) polymerase from the second subset of sites. Similarly
a repellant charge can be applied to interstitial regions of an
array that do not contain charge sensors in order to inhibit
polymerases from being transported to the interstitial regions or
to remove (e.g. via desorption or degradation) polymerases from the
interstitial regions.
In many configurations an amplifier that is used for a charge
sensor will occupy substantially more space than the sensor itself.
For example, readout circuitry may occupy an area of a detection
device that is anywhere from 2.times.2 microns to 20.times.20
microns, whereas a single nanowire transistor may occupy as little
as 100.times.500 nanometers. The size and dimensions of an array of
charge sensors can in some embodiments be limited by the space
occupied by multiple amplifiers, for example, if an amplifier is
present for each charge sensor. The limitation can be overcome in
particular embodiments of the present apparatus and methods by
configuring an array of charge sensors such that a each amplifier
is operationally connected to several charge sensors. For example,
2, 3, 4, 5, 6, 8, 10 or more charge sensors can be connected to the
same amplifier. Thus a higher density of charge sensors can be
present on an array than in a configuration where there is a one to
one connectivity between amplifiers and charge sensors. In
operation, an amplifier can be assigned to amplify signal from only
one of many charge sensors to which it is (or was at one time)
connected. For example, the array of charge sensors can be loaded
by contacting it with a fluid containing a plurality of reaction
components of a particular type. This loading technique may result
in a substantial number of the charge sensors being attached to
more than one reaction component of a particular type and others
not being loaded with any of that type of reaction component at
all. Among several charge sensors that are connected to a common
amplifier, a single sensor that has attached to only a single
reaction component can be distinguished from the others that are
overloaded or unloaded and the amplifier can be assigned to acquire
signal from the single loaded charge sensors while not acquiring
signal from the other charge sensors to which it is (or was)
connected.
In some embodiments, an individual charge sensor will have capacity
for greater than one reaction component of a particular type.
Taking a polymerase as an example, each charge sensor may have
capacity to attach several polymerase molecules at once. In such
cases, the polymerase can be attached to a repellant moiety that
occupies a volume of space that sterically hinders more than one of
the polymerases that is bound to another repellant moiety from
attaching to an individual charge sensor. Similarly, the repellant
moiety can have a charge polarity that electrostatically hinders
more than one of the polymerases that is bound to another repellant
moiety from attaching to the same charge sensor.
A particularly useful repellant moiety is nucleic acid. A repellant
nucleic acid can provide both steric and electrostatic hindrance to
limit occupancy. A repellant nucleic acid is well suited to
polymerases, nucleic acid enzymes and other reaction components
that have a binding affinity for nucleic acids. However, it will be
understood that this type of affinity is not necessary because
synthetic methods can be used to attach repellant nucleic acids to
reaction components, for example, using the linkers, tethers and
attachment chemistries set forth herein or known in the art. The
length, sequence composition, or secondary structure of the
repellant nucleic acid can be modulated to achieve a desired
occupancy. For example, larger nucleic acids can be used when the
charge sensors have a relatively high capacity for the reaction
component to which the charge sensor will be attached. Smaller
nucleic acids can be sufficient for limiting loading of smaller
charge sensors (or when relatively large reaction components or
highly charged reaction components are used thus requiring only a
small increase in repellant properties). Nucleic acids are also
useful as repellant moieties since the sequence of the nucleic acid
can be selected to achieve desired binding properties to a
polymerase or other nucleic acid enzyme.
In some embodiments, a repellant nucleic acid can be compacted into
a nanoball structure. Methods of compacting nucleic acids are known
in the art (for example, as described by Bloomfield, Curr. Opin.
Struct. Biol. 6(3): 334-41 (1996), and US Pat. App. Pub. No.
2007/0099208 A1, each of which is incorporated herein by reference
in its entirety). For example, an alcohol or polyamine such as
spermine or spermidine can be used. A compacted nucleic acid will
have a structure that is more densely packed than the structure of
the nucleic acid in the absence of a compacting agent or compacting
condition and the structure will typically resemble a ball or
globule. The generation of such compacted nucleic acid balls is
useful for creating repellant moieties. Various methods can be used
to generate balls of a desired size, for example, using various
compacting techniques and/or varying the number of copies in an
amplicon. Generally, the compacted amplicons have an average
diameter or width ranging from about 0.1 .mu.m to about 5 .mu.m,
for example, about 0.1 .mu.m, about 0.2 .mu.m, about 0.5 .mu.m,
about 1 .mu.m, 2 .mu.m, about 3 .mu.m, about 4 .mu.m and about 5
.mu.m.
Other polymeric molecules are also useful as repellant moieties,
including without limitation, polyethylene glycol, polythenes,
polypropylene, polyvinyl chloride, Teflon, nylon, polyamides,
polyacetals, polyesters, Buna rubbers, polyacrylates, polystyrene,
and polychlorotrifluoroethene. Beads or particles made of solid
support materials or gels can also function as repellant
moieties.
A repellant moiety can remain attached to a reaction component
while the reaction component participates in a particular reaction
that is to be detected by a charge sensor to which the reaction
component is attached. For example, a repellant moiety that is
bound to a polymerase while the polymerase is loaded onto and
attached to a charge sensor can remain attached to the polymerase
in a subsequent nucleotide addition reaction that is detected by
the charge sensor. Alternatively, the repellant moiety can be
removed from the reaction component after the reaction component
has been attached to a charge sensor. Taking again the example of a
polymerase, a repellant moiety such as repellant nucleic acid can
be removed from the polymerase prior to the polymerase
participating in a detected reaction with a target nucleic acid.
Repellant moieties that are bound to a polymerase or other reaction
component can be removed by techniques known to those skilled in
the art to result in removal. For example, non-covalently bound
moieties can be removed by washing, or competitive displacement
using other moieties that bind to the reaction component.
Covalently bound moieties can be removed by chemical or physical
means such as those set forth herein in regard to cleaving tethers.
Repellant moieties once removed from a reaction component can be
washed away from the charge sensor to which the reaction component
remains attached.
In some embodiments, a charge sensor that has a capacity for more
than one reaction component of a particular type, can be overloaded
such that an individual charge sensor is attached to several of the
reaction components and then reaction components can be removed (or
inactivated or degraded) from the charge sensor leaving only a
single active reaction component attached to the individual charge
sensor. For example, a method of attaching reaction components to
charge sensors can be carried out to create cleavable tethers
between each reaction component and the charge sensor to which it
is attached. In some cases the reaction components that are
delivered in a fluid can include precursors to cleavable tethers,
the charge sensors can include precursors to cleavable tethers or
both the reaction component and charge sensor can have precursors
that react together to form a cleavable tether.
Cleavable tethers can be cleaved by bond breakage due to physical
or chemical processes. For example, a method set forth herein can
include a step of cleaving a cleavable tether by photochemical
cleavage, electrochemical cleavage, electric field, mechanical
agitation, chemical cleavage or heat. Useful cleavable tethers and
their precursors include those used for modification of proteins
and are commercially available, for example, from Thermo Fisher
(Waltham, Mass.), or Sigma Aldrich (St. Louis, Mo.).
Other methods can be used to remove one or more reaction components
from a charge sensor. For example, removal can be achieved by
degrading one or more reaction components from each charge sensor.
Degradation can be achieved by physical methods such as heat,
photo-oxidation, sonication or the like. Chemical degradation is
also possible for example using pH changes, chemical denaturants,
proteases or the like. In some cases, the extent of degradation can
be modulated by contacting the sensor-attached reaction components
with a protection moiety. The amount of protection moiety supplied
to an array of charge sensors can be titrated to result in binding
to a single reaction component, on average, per charge sensor. When
degradation is subsequently carried out all but the protected
reaction component attached to each charge sensor will be degraded.
This will leave a single reaction component attached to each charge
sensor. The protection moiety can remain bound to the reaction
component as it participates in a reaction that is detected by the
charge sensor, or the protection moiety can be removed. In some
cases, the degradation can happen by binding the polymerase active
site with a chemically or photochemically active oligonucleotide
analog. A chemical or photochemical treatment can be applied to
crosslink the oligonucleotide analog to the polymerase. As a
result, these polymerase can be rendered incapable of accepting a
target nucleic acid, or primer, or the polymerase may be rendered
incapable of conformational changes (e.g. open-close conformation
changes) that would have been detected by charge sensor.
An example of a useful protection moiety is a nucleic acid such as
a DNA nanoball. For example, a nucleic acid can be used to bind to
a polymerase or other nucleic acid enzyme to provide stability
against denaturation or chemical modification. A nucleic acid can
also provide a steric block preventing proteases from having access
to a polymerase that is attached to a charge sensor. Another
example is a primer hybridized single stranded DNA; in this case
the 3' end of the primer will be bound to the catalytic center of
the polymerase, preventing binding of reactive polymerase
deactivation moieties. A further example of a protection moiety is
a protein such as an antibody that specifically targets a
polymerase. The protein or antibody can protect against chemical
modification or provide a steric block preventing proteases from
having access to the polymerase. If it is desirable to remove the
antibody, the antibody could be removed using, for example, heat.
An appropriate temperature would be one at which the antibody is no
longer stably bound to the polymerase, but at which the polymerase
is stable. Other materials that have been exemplified herein for
use as repellant moieties can serve as protection moieties.
Degradation, inactivation or removal of excess reaction components
from charge sensors can be carried out with or without monitoring
of the charge sensors to determine the extent of degradation or
removal. For example, in particular embodiments a process of
removing one or more reaction components of a particular type from
modified charge sensors can include steps of (i) removing one or
more of the reaction components from each of the modified charge
sensors, (ii) monitoring the charge sensors to distinguish the
presence of multiple reaction components from the presence of a
single reaction component, and (iii) discontinuing the removing to
leave a single one of the reaction components attached to each of
the modified charge sensors. The status of the charge sensor can be
monitored by detecting changes in signal from the charge sensor.
Alternatively or additionally, a different sensor can be used to
detect presence or absence of reaction components at the surface of
the charge sensor. Any of a variety of detection modalities can be
used including for example, fluorometry to detect fluorescent
labels on reaction components, optical scatter methods, absorbance
methods to detect chromophores and other analytical detection
methods known in the art pertaining to the detection of proteins
and other reaction components.
A method of the present disclosure can include a step of providing
one or more target nucleic acids to a solid support that comprises
at least one charge sensor. In particular embodiments the charge
sensor will have been previously attached to another reaction
component that will react with the nucleic acid(s) in a desired
reaction, examples of which include nucleic acid enzymes such as
polymerases. In other embodiments target nucleic acid(s) can be
delivered to a charge sensor before or at the same time that other
reaction components (e.g. nucleic acid enzymes or polymerases) are
delivered to the charge sensor(s).
Target nucleic acids used in a method or apparatus of the present
disclosure can be composed of DNA, RNA or analogs thereof. The
source of the target nucleic acids can be genomic DNA, messenger
RNA, or other nucleic acids from native sources. In some cases the
target nucleic acids that are derived from such sources can be
amplified prior to use in a method or composition herein. Any of a
variety of known amplification techniques can be used including,
but not limited to, polymerase chain reaction (PCR), rolling circle
amplification (RCA), multiple displacement amplification (MDA), or
random prime amplification (RPA). It will be understood that
amplification of target nucleic acids prior to use in a method or
apparatus set forth herein is optional. As such, target nucleic
acids will not be amplified prior to use in some embodiments of the
methods and compositions set forth herein. Target nucleic acids can
optionally be derived from synthetic libraries. Synthetic nucleic
acids can have native DNA or RNA compositions or can be analogs
thereof.
Exemplary biological samples from which target nucleic acids can be
derived include, for example, those from a mammal such as a rodent,
mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat,
cow, cat, dog, primate, human or non-human primate; a plant such as
Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or
soybean; an algae such as Chlamydomonas reinhardtii; a nematode
such as Caenorhabditis elegans; an insect such as Drosophila
melanogaster, mosquito, fruit fly, honey bee or spider; a fish such
as zebrafish; a reptile; an amphibian such as a frog or Xenopus
laevis; a Dictyostelium discoideum; a fungi such as Pneumocystis
carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or
Schizosaccharomyces pombe; or a Plasmodium falciparum. Target
nucleic acids can also be derived from a prokaryote such as a
bacterium, Escherichia coli, staphylococci or Mycoplasma
pneumoniae; an archae; a virus such as Hepatitis C virus, ebola
virus or human immunodeficiency virus; or a viroid. Target nucleic
acids can be derived from a homogeneous culture or population of
the above organisms or alternatively from a collection of several
different organisms, for example, in a community or ecosystem.
Target nucleic acids need not be derived from natural sources and
can instead be synthesized using known techniques. For example,
gene expression probes or genotyping probes can be synthesized and
used in the methods and apparatus set forth herein.
In some embodiments, target nucleic acids can be obtained as
fragments of one or more larger nucleic acids. Fragmentation can be
carried out using any of a variety of techniques known in the art
including, for example, nebulization, sonication, chemical
cleavage, enzymatic cleavage, or physical shearing. Fragmentation
may also result from use of a particular amplification technique
that produces amplicons by copying only a portion of a larger
nucleic acid. For example, PCR amplification produces fragments
having a size defined by the length of the nucleotide sequence on
the original template that is between the locations where flanking
primers hybridize during amplification.
A population of target nucleic acids, or amplicons thereof, can
have an average strand length that is desired or appropriate for a
particular application of the methods or apparatus set forth
herein. For example, the average strand length can be less than
about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides,
5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100
nucleotides, or 50 nucleotides. Alternatively or additionally, the
average strand length can be greater than about 10 nucleotides, 50
nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides,
5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or
100,000 nucleotides. The average strand length for a population of
target nucleic acids, or amplicons thereof, can be in a range
between a maximum and minimum value set forth above.
In some cases a population of target nucleic acids can be produced
under conditions or otherwise configured to have a maximum length
for its members. For example, the maximum length for the members
that are used in one or more steps of a method set forth herein or
that are present in a particular composition can be less than about
100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000
nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides or
50 nucleotides. Alternatively or additionally, a population of
target nucleic acids, or amplicons thereof, can be produced under
conditions or otherwise configured to have a minimum length for its
members. For example, the minimum length for the members that are
used in one or more steps of a method set forth herein or that are
present in a particular composition can be more than about 10
nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides,
1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000
nucleotides, or 100,000 nucleotides. The maximum and minimum strand
length for target nucleic acids in a population can be in a range
between a maximum and minimum value set forth above.
The present disclosure provides a method of detecting a nucleotide.
The method can include the steps of (a) providing a nucleotide
binding protein (e.g. a polymerase) tethered to a solid support
charge sensor; (b) providing one or more labeled nucleotides,
whereby the presence of the label can be detected by the charge
sensor when the label is in proximity to the charge sensor; and (c)
detecting binding of the labeled nucleotide to the protein using
the charge sensor.
The binding of a nucleotide to a nucleic acid binding enzyme, such
as a polymerase, can be detected based on the recruitment of a
charge label to the enzyme which in turn causes a detectable
perturbation in the field around the charge sensor to which the
enzyme is attached. Exemplary charge labels are set forth
previously herein. In particular embodiments, the charge label is
attached to the .beta.- or .gamma.-phosphate position of the
nucleotide. An advantage of attaching the label at the beta- or
gamma-phosphate position of the nucleotide is that the label can be
removed by the catalytic activity of polymerase when incorporating
the nucleotide into a nascent strand. However, the label need not
be removed by polymerase activity. Thus, the label can be attached
at any of a variety of positions on a nucleotide including for
example via a linker to the base moiety of a nucleotide (see, for
example, the nucleotide positions and linkers set forth in U.S.
Pat. Nos. 7,427,673; 7,414,116; and 7,057,026 and PCT publications
WO 91/06678 and WO 07/123744, each of which is incorporated herein
by reference in its entirety). A label can also be attached at the
alpha-phosphate position of the nucleotide or at the ribose moiety
of the nucleotide. A label attached to any of a variety of moieties
of a nucleotide can optionally be cleaved from the nucleotide after
being detected, for example, via cleavage of a charge linker.
A label used in a method or apparatus set forth herein can further
include an oligonucleotide moiety. Exemplary oligonucleotide
moieties include DNA, RNA, and PNA as set forth previously herein.
As exemplified previously herein, an oligonucleotide moiety can be
useful for hybridizing to a nucleic acid tether or other nucleic
acid so as to localize electric field perturbation to occur with a
desired distance of a charge sensor. The oligonucleotide moiety can
occur as an intermediary structure between the nucleotide and a
charged moiety. However, the charge moiety is optional and need not
be located at an end of the oligonucleotide moiety that is distal
to the point of attachment to the nucleotide. Although several
embodiments are exemplified herein with reference to nucleotide
analogs having oligonucleotide moieties that interact with nucleic
acid tethers, it will be understood that nucleotide analogs can
have other linker components in place of the oligonucleotide
moieties. The linker components can comprise one member of a
binding pair that interacts with another member that is part of a
tether,
In some embodiments, different nucleotide types can be attached to
different labels. Thus, the differences in signal arising from the
labels can be used to distinguish different nucleotide types. This
can be particularly useful for nucleic acid sequencing methods
where several different types of nucleotides are delivered to a
polymerase in a way that the several different types of nucleotides
are present in parallel during a detection event. For example, four
different nucleotides having four different charge moieties can be
used as exemplified previously herein with regard to FIG. 2 and
FIG. 3. Alternatively or additionally, the label moieties can
contain oligonucleotide moieties that hybridize to different tether
sequences to produce mutually distinct signals at a charge sensor.
In some embodiments several labeled nucleotides used in a method
set forth herein will have different charge labels, respectively,
but each of said labeled nucleotides will have an oligonucleotide
moiety that is capable of hybridizing to the same immobilized
tether sequence. As set forth previously herein, different
nucleotide analogs can have oligonucleotide moieties with different
lengths, or alternatively, two or more of the nucleotide analogs
can have oligonucleotide moieties that are the same length.
Different nucleotides need not have different labels. The different
nucleotides can be delivered to a reaction separately such that the
nucleotides are distinguished based on knowledge of when and where
they are delivered. For example, a sequencing reaction can include
sequential additions of four separate nucleotides per cycle with
washes between nucleotide additions. This separate delivery of
nucleotides can be done whether the different nucleotides are
uniquely labeled or uniformly labeled.
In particular embodiments, a method of nucleic acid sequencing can
be performed by (a) providing a polymerase tethered to a solid
support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting incorporation of the labeled nucleotide
into a nascent strand complementary to a template nucleic acid
using the charge sensor. A plurality of incorporation events can be
detected in succession to determine the sequence. Alternatively,
only a single incorporation event is detected for each nascent
strand and this information is combined with knowledge of the
sequence for the nascent strand (or the template to which it is
hybridized) to arrive at the sequence.
In multiplex embodiments, the solid support may include a plurality
of charge sensors that are tethered to polymerases, and the method
includes a step of detecting incorporation of a labeled nucleotide
into a nascent strand complementary to a template nucleic acid at
each polymerase in the plurality of polymerases. The plurality of
polymerases used in a multiplex embodiment can optionally include
at least two different types of polymerases. The different types of
polymerases can be selected to produce mutually distinguishable
signals detectable by the charge sensors when incorporating the
same type of nucleotide into a nascent strand of nucleic acid. In
this way, an array of charge sensors having different attached
polymerases (e.g. one per charge sensor) can distinguish a greater
variety of nucleic acid sequences or provide greater sensitivity
than would be available using an array having only one type of
polymerase attached to the charge sensors. For example, the same
template when sequenced by the action of two different polymerases
will produce two different series of signals. The two series of
signals can be compared or otherwise used in combination to provide
a more accurate nucleotide sequence than would be derivable from
only one series of signals from only one type of polymerase.
An array of the present disclosure, for example, having been
produced by a method set forth herein, can be used for any of a
variety of applications. A particularly useful application is
nucleic acid sequencing. One example is sequencing-by-synthesis
(SBS). In SBS, extension of a nucleic acid primer along a nucleic
acid template (e.g. a target nucleic acid or amplicon thereof) is
monitored to determine the sequence of nucleotides in the template.
The underlying chemical process can be polymerization (e.g. as
catalyzed by a polymerase enzyme). In a particular polymerase-based
SBS embodiment, nucleotides are added to a primer (thereby
extending the primer) in a template dependent fashion such that
detection of the order and type of nucleotides added to the primer
can be used to determine the sequence of the template. A plurality
of different templates at different charge sensors of an array set
forth herein can be subjected to an SBS technique under conditions
where events occurring for different templates can be distinguished
due to their location at a specific charge sensor of the array.
Flow cells provide a convenient format for housing an array that is
produced by the methods of the present disclosure and that is
subjected to an SBS or other detection technique that involves
repeated delivery of reagents in cycles. For example, to initiate a
first SBS cycle, one or more nucleotides (optionally having charge
labels), can be flowed into/through a flow cell that houses an
array of charge sensors each having an attached polymerase to which
a nucleic acid template is bound. Those sites of an array where
primer extension causes a nucleotide to be incorporated can be
detected. Optionally, the nucleotides can further include a
reversible termination property that terminates further primer
extension once a nucleotide has been added to a primer. For
example, a nucleotide analog having a reversible terminator moiety
can be added to a primer such that subsequent extension cannot
occur until a deblocking agent is delivered to remove the moiety.
Thus, for embodiments that use reversible termination, a deblocking
reagent can be delivered to the flow cell (before or after
detection occurs). Washes can be carried out between the various
delivery steps. The cycle can then be repeated n times to extend
the primer by n nucleotides, thereby detecting a sequence of length
n. Exemplary SBS procedures and fluidic systems that can be readily
adapted for use with an array produced by the methods of the
present disclosure are described, for example, in Bentley et al.,
Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO
91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414;
7,315,019; 7,405,281, and US 2008/0108082, each of which is
incorporated herein by reference in its entirety.
Sequencing-by-ligation reactions are also useful including, for
example, those described in Shendure et al. Science 309:1728-1732
(2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each of which is
incorporated herein by reference in its entirety. Some embodiments
can include sequencing-by-hybridization procedures as described,
for example, in Bains et al., Journal of Theoretical Biology
135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16,
54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and
WO 1989/10977, each of which is incorporated herein by reference in
its entirety. In both Sequencing-by-ligation and
sequencing-by-hybridization procedures, target nucleic acids (or
amplicons thereof) are subjected to repeated cycles of
oligonucleotide delivery and detection. Such methods can be readily
modified to detect ligase conformational changes or to detect
charge labeled oligonucleotides in place of the fluorescent
detection of optical labels described in the published methods.
Another useful application for an array of the present disclosure,
for example, having been produced by a method set forth herein, is
gene expression analysis. Gene expression can be detected or
quantified using RNA sequencing techniques, such as those, referred
to as digital RNA sequencing. RNA sequencing techniques can be
carried out using sequencing methodologies known in the art such as
those set forth above except that fluorescence detection of
optically labeled nucleotides can be replaced with the charge-based
detection methods set forth herein. Gene expression can also be
detected or quantified using hybridization techniques carried out
by direct hybridization to an array or using a multiplex assay, the
products of which are detected on an array. Exemplary molecular
biological assays that can be used for array-based expression and
genotyping analysis are described in U.S. Pat. Nos. 7,582,420;
6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos. 2005/0053980
A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which is
incorporated herein by reference in its entirety. These methods can
be readily adapted by replacing optical labels and fluorescence
detection with the charge-based detection techniques, and
optionally the charge labels, set forth herein.
A method of nucleic acid sequencing provided by the present
disclosure can include the steps of (a) providing a polymerase
tethered to a solid support charge sensor; (b) providing one or
more labeled nucleotides, whereby the presence of the label can be
detected by the charge sensor when the label is in proximity to the
charge sensor, wherein the one or more labeled nucleotides have
reversible terminator moieties; (c) detecting incorporation of the
one or more labeled nucleotides into a nascent strand complementary
to a template nucleic acid using the charge sensor, thereby forming
a reversibly terminated nascent strand; (d) modifying the
reversible terminated nascent strand to render the nascent strand
capable of further incorporation of nucleotide; and (e) repeating
(b) through (d) to obtain a sequence of the template nucleic
acid.
The use of reversibly terminated nucleotides in a sequencing
reaction provides advantages of step control to the polymerase
extension process that would otherwise be continuous. This step
control can be useful for increasing the amount of time that a
newly extended nucleic acid strand spends in a detectable state.
For example, a charge label that is on a reversibly terminated
nucleotide can be maintained on a nascent strand after being added
by a polymerase and until a desired amount of signal is accumulated
by the charge sensor. This can allow for increased signal
collection. Then the sequencing process can proceed by addition of
deblocking agent followed by subsequent cycles of nucleotide
addition.
Another advantage of the step control conferred by the use of
reversibly terminated nucleotides is the ability to synchronize an
ensemble of reaction components that undergo the same reaction.
Thus, a sequencing method set forth herein can be carried out for
multiple copies of the same nucleic acid bound to multiple
polymerases at the site where a charge sensor is located (e.g. the
multiple polymerases can be attached to a single charge sensor).
The detection step used to identify the nucleotide added during
each cycle of polymerase activity can be effectively synchronized
by use of reversibly terminated nucleotides. Although reversibly
terminated nucleotides provide advantages for ensemble detection,
it will be understood that sequencing methods employing reversibly
terminated nucleotides can be used when a single polymerase is
attached to an individual charge sensor.
An ensemble of sequencing reactions can be set up to include a
plurality of polymerases attached to a common charge sensor,
wherein the polymerases are bound to target nucleic acids having
the same template sequence and a primer (or nascent strand) with
the same sequence. The polymerases can be attached to the charge
sensor as set forth previously herein. Then one or more target
nucleic acids comprising multiple repeats of the same template
sequence can be contacted with the polymerases. For example, a
target nucleic acid molecule having concatameric repeats of the
template sequence can be delivered to the polymerases at the
particular site. In this case, the subunit of sequence that forms
each repeat can function as an individual template sequence.
Optionally, the nucleic acid encoding the concatameric repeat can
be cleaved to form individual molecules each having a single
template sequence. These individual molecules can then be sequenced
by polymerases at the site. The nucleic acid encoding the
concatameric repeat can be created by rolling circle amplification
(RCA), for example, as described in Lizardi et al., Nat. Genet.
19:225-232 (1998) and US Pat App. Pub. No. 2007/0099208 A1, each of
which is incorporated herein by reference in its entirety.
Also provided is a method of nucleic acid sequencing that includes
the steps of (a) providing a polymerase tethered to a solid support
charge sensor; (b) contacting the polymerase with a template
nucleic acid and one or more different nucleotide types under
conditions wherein the polymerase catalyzes addition of the one or
more nucleotide types to form a nucleic acid complement of the
nucleic acid template, and wherein the addition of one or more
different nucleotide types produces a conformational signal change
from the polymerase that is detected by the charge sensor; (c)
detecting a change in the signal from the polymerase using the
charge sensor; and (d) determining the rate, polarity, amplitude or
time duration for the change in the signal for the addition of the
one or more different nucleotide type, thereby determining a
sequence of nucleotides for the template nucleic acid.
In particular embodiments, a sequence of nucleotides for a nucleic
acid template can be determined based on conformational changes
occurring in a nucleic acid enzyme such as a polymerase.
Distinguishing the conformational changes that occur for each type
of nucleotide that the enzyme interacts with and determining the
sequence of those changes can be used to determine the sequence of
the nucleic acid. For example, a polymerase that sequentially adds
of nucleotides to a nascent nucleic acid strand undergoes
conformational changes with each nucleotide addition. As set forth
in further detail herein, the conformational changes that occur for
each type of nucleotide that is added can be distinguished using a
charge sensor (or based on knowledge of which nucleotide(s) is/are
fluidically delivered to the substrate where sequencing is being
monitored) and the sequence of those changes can be detected to
determine the sequence of the nucleic acid.
A nucleic acid enzyme can be labeled to produce one or more signals
indicative of a conformational change in the enzyme as it interacts
with one or more reactants such as a nucleic acid or nucleotide. A
polymerase can be conformationally labeled such that activity of
the polymerase can be monitored by detection of allosteric charge
movement. For example, a polymerase can be conformationally labeled
to allow detection of a signal indicative of nucleotide binding, a
signal indicative of addition of a nucleotide to a growing nucleic
acid molecule, or a signal indicative of an intermediate change in
the conformation of the polymerase between binding and catalysis.
Accordingly, a polymerase can include at least one non-natural
label moiety that is detected by the charge sensor. Polymerase can
be engineered to have negative and positive charges that maximize
the charge change per unit volume through allosteric movements. If
this unit volume is close to the charge sensor, this movement can
easily be detected. In particular embodiments, a signal detected by
a charge sensor from a conformationally labeled polymerase can
distinguish a binding event from a catalytic event. However, such a
distinction may not be necessary for some embodiments and the
signal can be merely indicative of the overall addition of a
nucleotide. Alternatively or additionally, the signal can
distinguish binding of a correctly base-paired nucleotide from
binding of an incorrectly base-paired nucleotide.
A particularly useful label moiety that can be attached to a
polymerase or other nucleic acid enzyme used in a method or
apparatus set forth herein is a negative charge label, examples of
which include, but are not limited to, a phosphate group, carboxyl
group, amino acid, DMT and/or FMOC. Also useful are positive charge
labels including, for example, a primary amine. Intrinsic labels
such as amino acid side chains or naturally occurring post
translational modifications (e.g. phosphorylation, addition of
flavin, reduction of disulfides or the like) can also provide
useful moieties for detection in a method or apparatus set forth
herein.
Some embodiments can employ a nucleotide analog that is
incorporated into a polynucleotide strand by a polymerase at a rate
that is measurably different than the rate at which another
nucleotide is incorporated into the strand by the polymerase.
Another useful nucleotide analog is one that is bound to a
polymerase at a rate that is measurably different than the rate at
which another nucleotide is bound to the polymerase. A nucleotide
analog that causes a conformational change of a polymerase at a
rate that is measurably different than another nucleotide is also
useful. The relative rate of binding, incorporation or polymerase
conformational change for a nucleotide analog can be measured
relative to a natural nucleotide having the same Watson-Crick base
pairing partner or relative to other nucleotides that are used in a
nucleic acid synthesis reaction. The relative rate can be faster or
slower for the nucleotide analog.
According to particular embodiments, a polymerase or other nucleic
acid enzyme can be conformationally labeled. Conformational
labeling of nucleic acid enzymes provides advantages for nucleic
acid sequence analysis. Conformationally labeled molecules, and
methods for making and using them, will be exemplified below with
regard to labeled polymerases. It will be understood that other
nucleic acid enzymes such as exonucleases and reverse
transcriptases can be made and used similarly.
Polymerases undergo conformational changes in the course of
synthesizing a nucleic acid polymer. For example, polymerases
undergo a conformational change from an open conformation to a
closed conformation upon binding of a nucleotide. Thus, a
polymerase that is bound to a nucleic acid template and growing
primer is in what is referred to in the art as an "open"
conformation. A polymerase that is bound to a nucleic acid
template, primer and a correctly base paired nucleotide is in what
is referred to in the art as a "closed" conformation. At a more
detailed structural level, the transition from the open to closed
conformation is characterized by relative movement within the
polymerase resulting in the "thumb" domain and "fingers" domain
being closer to each other. In the open conformation the thumb
domain is further from the fingers domain, akin to the opening and
closing of the palm of a hand. In various polymerases, the distance
between the tip of the finger and the thumb can change up to 30
angstroms between the "open" and "closed" conformations. The
distance between the tip of the finger and the rest of the protein
domains can also change up to 10 angstroms. It will be understood
that larger changes may also occur and can be exploited in a method
set forth herein such that a change that is greater than 10
angstroms can be detected. Furthermore, smaller changes can be
detected including those that are less than about 10, 8, 6, 4, or 2
angstroms so long as the change in distance is sufficient to be
detectable using a charge sensor.
In particular embodiments, a charge label that is attached to a
finger domain can be attached to a residue at position 376 or
residues within 5 angstroms radius from position 376 of the Phi29
DNA polymerase and a label that is attached to the thumb or other
domain can be attached to a residue at position 535, 203, 510, 564,
or residues within 5 an